KR102310775B1 - Pharmaceutical nanoparticles showing improved mucosal transport - Google Patents

Abstract

Compositions with improved particle transport in mucus are provided. In some embodiments, the compositions and methods comprise preparing mucus-penetrating particles (MPPs) without any polymeric carrier or with minimal use of polymeric carriers. The composition may comprise modifying the surface coating of the particles formed with the medicament having low water solubility. Such compositions can be used to achieve efficient transport of particles of a drug across the mucus barrier in the body for a wide range of applications including drug delivery, imaging and diagnostic applications.

Description

Pharmaceutical nanoparticles exhibiting improved mucosal transport {PHARMACEUTICAL NANOPARTICLES SHOWING IMPROVED MUCOSAL TRANSPORT}

Related applications

This application is entitled "Nanocrystals, Compositions, and Methods to Aid Particle Transport in Mucus" to 35 U.S.C. Priority under § 119(e), this provisional application is incorporated herein by reference.

field of invention

The present invention relates generally to nanocrystals, compositions and methods for assisting particle transport in mucus.

The mucus layer, present at various points of entry into the body, including the eyes, nose, lungs, gastrointestinal tract, and female reproductive system, is naturally sticky, effectively trapping pathogens, allergens and debris and preventing mucus turnover. It serves to protect the body against them by rapidly removing them. For effective delivery of therapeutic, diagnostic or imaging particles across mucous membranes, the particles must be able to easily penetrate the mucus layer to avoid mucus adhesion and rapid mucus clearance. Some types of evidence suggest that conventional nanoparticles cannot cross the mucosal barrier. However, polymer nanoparticles (whether degradable or not) modified (covalently or non-covalently) with special surface coatings have recently been shown to be able to diffuse in physiologically rich mucus samples as rapidly as they do in water. has been proven in These polymer-based mucus-penetrating particles (MPPs) can encapsulate a variety of therapeutic, imaging or diagnostic agents to enable drug delivery, diagnostic or imaging applications.

Nevertheless, polymer-based MPPs may have some inherent limitations compared to unencapsulated particles of pharmaceutical agents. In particular, given drug delivery applications, these limitations may include: 1) inherently lower drug loading; 2) a less convenient dosage form for the polymer nanoparticles as recovery from dry powder storage form may be required; 3) potentially increased toxicity; 4) chemical and physical stability concerns; and 5) increased manufacturing complexity. Accordingly, improvements in compositions and methods comprising mucus-penetrating particles for the delivery of medicaments would be beneficial.

Summary of invention

The present description relates generally to nanocrystals, compositions and methods that aid in particle transport in mucus. In some embodiments, the compositions and methods include mucus-penetrating particles without any polymeric carrier, or with minimal use of a polymeric carrier. The subject matter of this application, in some cases, includes interrelated products, alternative solutions to particular problems, and/or many different uses of structures and compositions.

In one set of embodiments, a method of forming coated particles is provided. The method comprises combining the core particle with a solution comprising a surface-altering agent, wherein the core particle comprises a solid drug or a salt thereof, wherein the drug or salt is about 1 mg in solution at 25°C. /mL or less, wherein the drug or a salt thereof constitutes at least about 80 wt% of each of the core particles. The method also comprises coating the core particle with a surface-altering agent to form coated particles, wherein the surface-altering agent comprises a triblock copolymer comprising a hydrophilic block-hydrophobic block-hydrophilic block configuration, wherein the hydrophobic block comprises: having a molecular weight of at least about 2 kDa, wherein the hydrophilic block constitutes at least about 15 wt % of the triblock copolymer, wherein the hydrophobic block is associated with the surface of the core particle, wherein the hydrophilic block is present on the surface of the coated particle to provide the coating renders the coated particles hydrophilic, wherein the coated particles have a relative velocity greater than 0.5 in mucus.

In another set of embodiments, a composition comprising a plurality of coated particles is provided. The coated particle comprises a core particle comprising a solid drug or salt thereof, wherein the drug or salt has an aqueous solubility of about 1 mg/mL or less at 25°C at any point throughout the pH range and , wherein the drug or salt thereof constitutes at least about 80 wt % of the core particle. The coated particles also comprise a coating comprising a surface modifier surrounding the core particle, wherein the surface modifier comprises a triblock copolymer comprising a hydrophilic block-hydrophobic block-hydrophilic block configuration, wherein the hydrophobic block has a molecular weight of at least about 2 kDa, and the hydrophilic block constitutes at least about 15 wt % of the triblock copolymer, wherein the hydrophobic block is associated with the surface of the core particle, wherein the hydrophilic block is present on the surface of the coated particle, renders the coated particle hydrophilic, wherein the surface-altering agent is present on the surface of the core particle at a density of at least about 0.001 molecules per nanometer squared. The coated particles have a relative velocity greater than 0.5 in mucus.

In another set of embodiments, a method of forming coated particles comprises providing a medicament and precipitating the medicament by forming a salt in an aqueous solution in the presence of a surface-altering agent to form core particles of the medicament, wherein the salt Silver has a lower water solubility than the non-salt form of the drug, and the water solubility of the salt is about 1 mg/mL or less at 25° C. at any point throughout the pH range, wherein the surface-altering agent is about 0.01% in aqueous solution ( w/v) or higher. The method comprises coating the core particle with a surface-altering agent to form coated particles, wherein the surface-altering agent comprises a triblock copolymer comprising a hydrophilic block-hydrophobic block-hydrophilic block configuration, wherein the hydrophobic block is about having a molecular weight of at least 2 kDa, wherein the hydrophilic block constitutes at least about 15 wt % of the triblock copolymer, wherein the hydrophobic block is associated with the surface of the core particle, wherein the hydrophilic block is present on the surface of the coated particle to be coated Make the particles hydrophilic. The coated particles have a relative velocity greater than 0.5 in mucus.

In another set of embodiments, a method of treatment is provided. The method comprises administering to a patient or subject in need thereof a composition comprising a plurality of coated particles. The coated particles comprise core particles comprising a solid drug or a salt thereof, wherein the drug or salt has a solubility in water at 25° C. of no more than about 1 mg/mL at any point throughout the pH range, wherein the drug or salt Their salts constitute at least about 80 wt % of the core particles. The core particle also comprises a coating comprising a surface modifier surrounding the core particle, wherein the surface modifier comprises a triblock copolymer comprising a hydrophilic block-hydrophobic block-hydrophilic block configuration, wherein the hydrophobic block comprises: having a molecular weight of at least about 2 kDa, wherein the hydrophilic block constitutes at least about 15 wt % of the triblock copolymer, wherein the hydrophobic block is associated with the surface of the core particle, wherein the hydrophilic block is present on the surface of the coated particle to provide the coating made the particles hydrophilic, wherein the surface-altering agent is present on the surface of the core particle at a density of at least about 0.001 molecules per nanometer squared. The coated particles have a relative velocity greater than 0.5 in mucus.

Other advantages and novel features of the invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying drawings. In the event that this specification and documents incorporated by reference contain conflicting and/or inconsistent disclosures, the present specification shall control. If two or more documents incorporated by reference contain contradictory and/or inconsistent disclosures with respect to each other, the later effective date shall prevail.

BRIEF DESCRIPTION OF THE DRAWINGS Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying drawings, which are schematic and not drawn to scale. In the drawings, each identical or nearly identical component exemplified is typically represented by a single number. For purposes of clarity, not every component is depicted in every figure, nor is every component of each embodiment of the invention depicted unless the illustration is essential to an understanding of the invention by one of ordinary skill in the art. In the drawing,
1 is a schematic diagram of a mucus-penetrating particle having a coating and a core of solid medicament, according to one set of embodiments.
2A shows 200 nm carboxylated polystyrene particles (negative control), 200 nm PEGylated polystyrene particles (positive control), and prepared by nanomilling and coated with different stabilizers/surface modifiers, according to one set of embodiments. Plot showing _{ensemble averaged velocity <V mean} > in human cervical mucus (CVM) for nanocrystalline particles (sample).
2B is a plot showing _{the relative velocity <V mean} > _relative in CVM for nanocrystal particles prepared by nanomilling and coated with different stabilizers/surface modifiers, according to one set of embodiments.
Figure 3A-3D, the trajectory of the CVM within the ensemble of the coated nano-crystal grains different surface modification agent according to one set of embodiments - is a histogram showing the mean speed (trajectory-mean velocity) V _mean of _the distribution.
4 is nanocrystal particles coated with ^{different PEO-PPO-PEO Pluronic ®} triblock copolymers, mapped to molecular weight and PEO weight content (%) of PPO blocks, according to one set of embodiments. A plot showing the _relative <V _mean > in CVM for .
^{5 is bulk transport via CVM for solid particles with different core materials coated with either Pluronic ®} F127 (MPP) or sodium dodecyl sulfate (CP, negative control), according to one set of embodiments. (mass transport) is a plot.
6A-6C show the eyelids of New Zealand white rabbits after administration of particles of loteprednol etabonate coated with prescription loteprednol etabonate, lotemax®, or pluronic® F127, according to one set of embodiments. Drug levels of loteprednol etabonate in the conjunctiva ( FIG. 6A ), ocular conjunctiva ( FIG. 6B ), and cornea ( FIG. 6C ) are shown.
7A and 7B are physicochemical characterizations of CUR-1% F127 particles according to one set of embodiments. 7A is a powder X-ray diffraction (Powder-XRD) diagram of F127, raw curcumin and CUR-1% F127 particles. 7B is a representative transmission electron microscope (TEM) image of CUR-1% F127 particles.
8A and 8B show CUR-1% F127 particles, 200 nm in CVM ( FIG. 8A ) and human cystic fibrosis sputum (CFS) ( FIG. 8B ) as a function of time scale, according to one set of embodiments. ensemble averaged geometric mean-square displacement (<MSD>) of carboxylated polystyrene (PSCOOH) and 200 nm PEGylated polystyrene (PSPEG) particles. Data represent the ensemble mean of 5 independent experiments, with n ≥ 100 for each experiment. Error bars represent geometric standard error.
9 is a plot showing geometric ensemble effective diffusivity (<Deff>) at a time scale of 1 s for CUR particles formulated with different concentrations of F127 in human CVM, according to one set of embodiments. Data represent the ensemble mean of at least 3 independent experiments, with n ≥ 100 for each experiment. Error bars represent geometric standard error.
10A-10C are plots showing the diffusivity of CUR particles formulated with ^{different Pluronics ®} in human CVM, according to one set of embodiments. Figure 10A shows a fluorenyl Optronics ^® poly (ethylene glycol) (PEG) segments and poly (propylene oxide) (PPO) Distribution of <Deff> in the time scale of 1 s with respect to the molecular weight (MW) of the segments. Each data point represents a specific type ^{of Pluronics ® .} PPO and PEG MW were evaluated based on the molecular weight provided by the manufacturer. 10B-C show <Deff> on a time scale of 1 s as a function of MW of (B) PEG or (C) PPO segments. The inset shows the same plot using a linear scale of <Deff>, while R represents the correlation coefficient. Data represent the ensemble mean of at least 3 independent experiments, with n ≥ 100 for each experiment. Error bars represent geometric standard error.
11 is a plot showing the cumulative release of CUR-1% F127 particles in phosphate buffered saline (pH=7.4) using octanol extraction, according to one set of embodiments.
12 is a plot showing the cumulative release of free TFV in solution versus TFV-Zn particles in suspension from a dialysis bag to conventional phosphate buffered saline, according to one set of embodiments.
13A-13B show muco-penetrating / F127-coated TFV particles (FIG. 13A) and muco-adhesive (muco) on flattened vaginal tissue from human-like estrus phase mice, according to one set of embodiments. -adhesive) / is an image showing the distribution of PVA-coated TFV particles (Fig. 13B). Vaginal tissue was removed within 10 minutes of administration.
14A-14D show the transport rates of CP and MPP in mouse estrous CVM. (FIG. 14A) Representative trajectories of particles exhibiting effective diffusivity within one SEM of the ensemble mean at a time scale of 1 s. ( FIG. 14B ) Ensemble-averaged geometric mean square displacement (<MSD>) as a function of time scale. Data on particles on ex vivo mouse vaginal tissue (mCVM) compared to identical particles in ex vivo human CVM (hCVM) (SK Lai, YY Wang, K. Hida, R. Cone, J. Hanes, nanoparticles reveal that human cervicovaginal mucus is riddled with pores larger than viruses. P Natl Acad Sci USA 107, 598-603 (2010)]) and the theoretical diffusion rate of 110 nm particles in water (~4 μm/s) . (Fig. 14C) Distribution of logarithms of individual particle effective diffusivity (D _{eff ) on a time scale of 1 s.} Data represent the ensemble mean of three independent experiments, with n ≥ 150 particles for each experiment. The diffusivity values to the left of the dotted line indicate particles with MSD values smaller than the particle diameter. (FIG. 14D) Percentage of particles capable of penetrating a 100 μm-thick layer of mouse CVM over time, based on Fick's Second Law of diffusion simulation of particles undergoing random diffusion , where the diffusivity is equivalent to the experimentally measured diffusivity of the particle. 14E graphically depicts vaginal drug delivery from gel, CP, and MPP formulations.
15A-15B are plots showing the transport of nanoparticles on IE mouse vaginal tissue. Ensemble-averaged geometric mean square displacement (<MDS>) as a function of time scale. ( FIG. 15A ) Data regarding MPP and CP on ex vivo vaginal tissues of induced estrus (IE) and spontaneous cycle estrus mice. (FIG. 15B) Biodegradable MPP on IE tissue was compared to non-biodegradable MPP. Data represent ensemble means of at least 3 independent experiments, with mean n > 150 particles and at least 130 particles for each experiment. Data are presented as means with standard error of the mean (SEM).
16 includes images illustrating particle distribution in the vagina of a mouse. Distribution of red fluorescent non-biodegradable and biodegradable CP and MPP in transverse cryosections of vaginal tissue from estrous and IE mice. Images are representative of n ≥ 3 mice.
17 includes images and plots showing quantification of vaginal nanoparticle coverage. Distribution of red fluorescent non-biodegradable and biodegradable CP and MPP on flattened estrous mouse vaginal tissue. Insets are images of dark areas at higher magnifications. Images are representative of averages calculated for n ≥ 3 mice. Data are mean ± SEM. *P < 0.05, Student's t test.
18 includes images showing cervical nanoparticle coverage. Cervical distribution of red fluorescent non-biodegradable and biodegradable CP and MPP on cervical tissue of estrus mice. Insets are images of dark areas at higher magnifications. Images are representative of n ≥ 3 mice. Data are mean ± SEM. *P < 0.05, Student's t test.
19 includes images showing the effect of mucus removal on mucoadhesive nanoparticles. Red fluorescence in transverse cryosections of mouse vaginal tissue with either an intact mucus layer (untreated) or mucus removed by lavage and swabbing (mucilage removed). Distribution of degradable and biodegradable CPs. Images are representative of n ≥ 3 mice.
20 includes images showing particle distribution in the vagina of IE mice. Distribution of red fluorescent non-biodegradable CP and MPP in transverse cryosections of IE mouse vaginal tissue. Images are representative of n ≥ 3 mice.
21A-21B are images and plots showing retention of non-biodegradable MPP and CP in IE mouse cervical tract. (FIG. 21A) Overlay of particle fluorescence intensity and bright-field images for CP and MPP in whole cervical tissue. ( FIG. 21B ) Fraction of particles remaining over time based on quantification of particle fluorescence. Data are mean ± SEM (n ≥ 7). *P < 0.05, Student's t test.
22 includes images showing the distribution and retention of a model drug, FITC, in the vagina of estrus mice encapsulated in biodegradable MPP or delivered in gel form. After 24 h, a fluorescence image of the smoothed mouse vaginal tissue was taken. Images are representative of n ≥ 3 mice. Data are mean ± SEM. *P < 0.05, Student's t test.
23 includes images showing acute toxicity and cytokine concentration administered daily. Hematoxylin and eosin (H&E)-stained cross-sections of excised mouse DP vaginal tissue 24 h after vaginal administration of 5% N9, PBS, CP, MPP, BD-CP, and BD-MPP. A scale bar is applied to all images. Arrowheads indicate clusters of neutrophils. Images are representative of n ≥ 5 mice.
24 is a plot showing the cytokine concentration administered daily. Cytokine concentrations in DP mouse cervical lavage (CVL) after daily vaginal treatment for 7 days. Data are mean ± SEM. *P < 0.05, Student's t test.
25 is a bar graph showing the relationship between the relative velocity of polystyrene particles coated with Pluronic® F127 in mucus and the density of Pluronic® F127 molecules on the particle surface.

details

Nanocrystals, compositions and methods are provided to aid particle transport in mucus. In some embodiments, the compositions and methods comprise preparing mucus-penetrating particles (MPPs) without any polymeric carrier or with minimal use of polymeric carriers. The compositions and methods, in some embodiments, may include modifying the surface coating of particles formed with a medicament having low water/aqueous solubility. Such methods and compositions can be used to achieve efficient transport of particles of a drug across the mucus barrier in the body for a wide range of applications including drug delivery, imaging and diagnostic applications. In certain embodiments, pharmaceutical compositions comprising such particles are suitable for routes of administration involving particles that cross the mucosal barrier.

In some embodiments, the particles described herein have a core-shell type arrangement. The core may comprise a solid drug or a salt thereof having a relatively low water solubility. The core may be coated with a coating or shell comprising a surface-altering agent that promotes the mobility of the particles in mucus. As described in more detail below, in some embodiments the surface modifier may comprise a triblock copolymer comprising a hydrophilic block-hydrophobic block-hydrophilic block configuration. The molecular weight of each of the hydrophilic and hydrophobic blocks can be selected to impart specific transport characteristics to the particle, such as increased transport through mucus.

Non-limiting examples of particles are now provided. As shown in the exemplary embodiment of FIG. 1 , particle 10 includes a core 16 (which may be in the form of a particle, referred to herein as a core particle) and a coating 20 surrounding the core. do. In one set of embodiments, a substantial portion of the core is formed of one or more solid agents (eg, drugs, therapeutic agents, diagnostic agents, imaging agents) capable of causing certain beneficial and/or therapeutic effects. The core can be, for example, nanocrystals (ie, nanocrystalline particles) of the drug. The core includes a surface 24 to which one or more surface-altering agents may be adhered. For example, in some cases, the core 16 is surrounded by a coating 20 comprising an inner surface 28 and an outer surface 32 . The coating may be formed, at least in part, of one or more surface-altering agents 34 , such as polymers (eg, block copolymers), capable of associating with the surface 24 of the core. The surface modifier 34 may be, for example, covalently attached to the core particle, non-covalently attached to the core particle, adsorbed to the core, or ionic interaction, hydrophobic and/or hydrophilic interaction, electrostatic interaction, van der It may associate with the core particle by attaching to the core through a Val's interaction, or a combination thereof. In one set of embodiments, the surface-altering agent, or portion thereof, is selected to facilitate transport of the particle through a mucosal barrier (eg, mucus or mucosa).

In certain embodiments described herein, the one or more surface-altering agents 34 are oriented in a particular configuration to the coating of particles. For example, in some embodiments where the surface modifier is a triblock copolymer, such as a triblock copolymer having a hydrophilic block-hydrophobic block-hydrophilic block configuration, the hydrophobic block 36 can be oriented towards the surface of the core, and Block 38 may be oriented away from the core surface (eg, towards the outside of the particle). The hydrophilic block may have characteristics that facilitate transport of particles across the mucosal barrier, as described in more detail below.

Particle 10 may optionally include one or more components 40 , such as, optionally, a targeting moiety, a protein, a nucleic acid, and a bioactive agent that may confer specificity to the particle. For example, a targeting agent or molecule (eg, a protein, nucleic acid, nucleic acid analog, carbohydrate, or small molecule), if present, can aid in directing the particle to a particular location in the subject's body. The location can be, for example, a tissue, a particular cell type, or an intracellular compartment. One or more components 40, if present, may be associated with the core, the coating, or both; For example, they may associate with the surface 24 of the core, the inner surface 28 of the coating, the outer surface 32 of the coating and/or be embedded in the coating. The one or more components 40 may be associated through covalent bonding, absorption, or attached through ionic interactions, hydrophobic and/or hydrophilic interactions, electrostatic interactions, van der Waals interactions, or combinations thereof. In some embodiments, the component may be attached (eg, covalently) to one or more surface-altering agents of the coated particle using methods known to those of skill in the art.

It should be understood that components and configurations other than those shown in FIG. 1 or described herein may be suitable for particular particles and compositions, and in some embodiments not all of the components shown in FIG. 1 are necessarily present.

In one set of embodiments, the particle 10, upon introduction into the subject, is one or more components, such as mucus, cells, tissues, organs, particles, fluids (eg, blood), portions thereof, and combinations thereof, in the subject. can interact with In some such embodiments, the coating of coated particles 10 comprises a surface-altering agent or other component having properties that allow for beneficial interaction (eg, transport, binding, adsorption) with one or more substances from a subject. can be designed to For example, the coating may include a surface-altering agent or other component having a particular hydrophilicity, hydrophobicity, surface charge, functional group, specificity for binding, and/or density, which promotes or reduces a particular interaction in a subject. can One specific example is by selecting a particular hydrophilicity, hydrophobicity, surface charge, functional group, specificity for binding, and/or density of one or more surface-altering agents that reduce physical and/or chemical interactions between the particles and the mucus of the subject. , to improve the mobility of particles through mucus. Other examples are described in more detail below.

In some embodiments, once a particle is successfully transported across a mucosal barrier (eg, mucus or mucosa) in a subject, further interactions between the particles may occur in the subject. The interaction may, in some cases, occur through the coating and/or core, for example, from one or more components of a subject to particle 10, and/or from particle 10 to one or more components of a subject. exchange of substances (eg, pharmaceuticals, therapeutics, proteins, peptides, polypeptides, nucleic acids, nutrients). For example, in some embodiments where the core is formed of or comprises a drug, degradation, release, and/or transport of the drug from the particle may result in certain beneficial and/or therapeutic effects in the subject. As such, the particles described herein may be used in the diagnosis, prevention, treatment or management of a particular disease or physical condition.

Specific examples of the use of the particles described herein are provided below in the context of being suitable for administration to a mucosal barrier (eg, mucus or mucosa) in a subject. Although many embodiments herein are described in this context and in the context of providing benefits to diseases and conditions associated with the transport of substances across mucosal barriers, the invention is not so limited and the particles, compositions, and compositions described herein are not so limited. It should be understood that the , kits, and methods can be used to prevent, treat, or manage other diseases or physical conditions.

Mucus is a viscous, viscoelastic gel that protects against pathogens, toxins and foreign bodies at various points of entry into the body, including the eyes, nose, lungs, gastrointestinal tract and female reproductive system. Many synthetic nanoparticles are strongly mucoadhesive and are effectively entrapped in the rapidly cleared surrounding mucus layer, which greatly limits their distribution throughout the mucosa as well as penetration towards the underlying tissues. The residence time of these entrapped particles is limited by the rate of replacement of the surrounding mucus layer, which ranges from seconds to hours, depending on the organ. To ensure effective delivery of particles, including pharmaceuticals (eg, therapeutic, diagnostic, and/or imaging agents) across mucous membranes, such particles should readily diffuse through the mucus barrier, avoiding mucus adhesion.

It has recently been demonstrated that modifying the surface of polymeric nanoparticles with a mucus-penetrating coating can minimize adhesion to mucus and thus enable rapid particle penetration across the mucus barrier. Specifically, polymer nanoparticles as small as 500 nm in size covalently with a dense coating of low molecular weight PEG (2 kDa -5 kDa) or certain Pluronic ^® molecules (eg P103, P105, F127) It has been found that when coated non-covalently with mucous membranes, they can penetrate human mucus almost as quickly as they migrate in pure water, and nearly 100 times faster than similar-sized uncoated polymer particles.

Nevertheless, polymer-based mucus-penetrating particles may have one or more inherent limitations in some embodiments. In particular, given drug delivery applications, these limitations may include one or more of the following: A) low drug encapsulation efficiency and low drug loading: The encapsulation of a drug into polymeric particles is usually due to the encapsulation of the drug into the particles during manufacture. Less than 10% of the total amount of drug used, often ineffective. Moreover, drug loadings greater than 50% are seldom achieved. B) Ease of Use: Formulations based on drug-loaded polymer particles should generally, typically, be stored as a dry powder to avoid premature drug release, and thus be subject to point-of-use restoration ( re-constitution) or a high-performance dosing device. C) Biocompatibility: The accumulation of slowly degrading polymer carriers after repeated administration and their toxicity over a long period of time exist as major concerns for polymeric drug carriers. D) Chemical and Physical Stability: Polymer degradation can impair the stability of the encapsulated drug. In many encapsulation processes, the drug undergoes a transition from solution phase to solid phase, which is not well controlled in terms of the physical form of the emerging solid phase (ie, amorphous versus crystalline versus crystalline polymorph). This is a concern for multiple aspects of formulation performance, including physical and chemical stability and release kinetics. E) Manufacturing complexity: Preparation of drug-loaded polymer MPPs, particularly scalability, is a fairly complex process that typically involves multiple steps and significant amounts of toxic organic solvents.

In some embodiments described herein, compositions and methods of making particles, including certain compositions and methods for making particles with increased transport across mucosal barriers, address one or more, or all, of the concerns described above. Specifically, in some embodiments, the compositions and methods do not include encapsulation with a polymeric carrier or involve minimal use of a polymeric carrier. Advantageously, by avoiding or minimizing the need to encapsulate a pharmaceutical agent (eg, drug, imaging agent, or diagnostic agent) in a polymeric carrier, drug loading, ease of use, biocompatibility, stability, and/or complexity of manufacturing are reduced. Certain limitations of polymer MPPs can be addressed. The methods and compositions described herein can facilitate clinical development of mucus-penetrating particle technology.

core particle

As described above with reference to FIG. 1 , the particle 10 may include a core 16 . The core may be formed of any suitable material, such as an organic material, an inorganic material, a polymer, or a combination thereof. In one set of embodiments, the core comprises a solid. The solid can be, for example, a crystalline or amorphous solid, such as a crystalline or amorphous solid pharmaceutical (eg, therapeutic, diagnostic, and/or imaging agent), or a salt thereof. In some embodiments, more than one agent may be present in the core. Specific examples of medicaments are provided in more detail below.

The agent may be present in any suitable amount, for example, about 1 wt% or more, about 5 wt% or more, about 10 wt% or more, about 20 wt% or more, about 30 wt% or more, about 40 wt% or more, about 50 wt% or more of the core. at least about 60 wt%, at least about 70 wt%, at least about 80 wt%, at least about 85 wt%, at least about 90 wt%, at least about 95 wt%, or at least about 99 wt%, may exist. In one embodiment, the core is formed of 100% drug. In some cases, the agent is about 100 wt% or less, about 90 wt% or less, about 80 wt% or less, about 70 wt% or less, about 60 wt% or less, about 50 wt% or less, about 40 wt% or less, about 30 wt% or less wt % or less, about 20 wt % or less, about 10 wt % or less, about 5 wt % or less, about 2 wt % or less, or about 1 wt % or less in the core. Combinations of the aforementioned ranges are also possible (eg, present in an amount greater than or equal to about 80 wt % and less than or equal to about 100 wt %). Other ranges are also possible.

In embodiments where the core particle comprises a relatively large amount of drug (e.g., at least about 50 wt % of the core particle), the core particle generally provides an increased amount of drug as compared to particles formed by encapsulating the agent in a polymeric carrier. have loading. This is an advantage for drug delivery applications, since higher drug loading means that fewer particles may be needed to achieve the desired effect compared to the use of particles containing a polymeric carrier.

In some embodiments, the core may be formed of a solid material having a relatively low water solubility (ie, solubility in water, optionally with one or more buffers), and/or a relatively low solubility in a solution in which the solid material is coated with a surface-altering agent. can For example, the solid material may be about 5 mg/mL or less, about 2 mg/mL or less, about 1 mg/mL or less, about 0.5 mg/mL or less, about 0.1 mg/mL or less, about 0.05 mg/mL or less at 25°C. or less, about 0.01 mg/mL or less, about 1 μg/mL, about 0.1 μg/mL or less, about 0.01 μg/mL or less, about 1 ng/mL or less, about 0.1 ng/mL or less, or about 0.01 ng/mL or less of water solubility (or solubility in coating solution). In some embodiments, the solid material is at least about 1 pg/mL, at least about 10 pg/mL, at least about 0.1 ng/mL, at least about 1 ng/mL, at least about 10 ng/mL, at least about 0.1 μg/mL, about 1 μg/mL or more, about 5 μg/mL or more, about 0.01 mg/mL or more, about 0.05 mg/mL or more, about 0.1 mg/mL or more, about 0.5 mg/mL or more, about 1.0 mg/mL or more, about It may have a solubility in water (or solubility in coating solution) of 2 mg/mL or greater. Combinations of the above-mentioned ranges are possible (eg, a solubility in water or a solubility in a coating solution of about 10 pg/mL or more and about 1 mg/mL or less). Other ranges are also possible. A solid material may have water solubility in these or other ranges at any point throughout the pH range (eg, pH 1 to pH 14).

In some embodiments, the core may be formed of a material within one of the ranges of solubility classified by the U.S. Pharmacopeia Convention: e.g., very soluble: >1,000 mg/mL; freely soluble: 100-1,000 mg/mL; Soluble: 33-100 mg/mL; sparing soluble: 10-33 mg/mL; Slightly soluble: 1-10 mg/mL; very slightly soluble: 0.1-1 mg/mL; and practically insoluble: <0.1 mg/mL.

Although the core is hydrophobic or hydrophilic, in many embodiments described herein, the core is substantially hydrophobic. "Hydrophobic" and "hydrophilic" are given their ordinary meanings in the art and, as will be understood by one of ordinary skill in the art, are, in many instances herein, relative terms. The relative hydrophobicity and hydrophilicity of a material can be determined by measuring the contact angle of a water droplet on the plane of the material being measured, using, for example, an instrument such as a contact goniometer and a packed powder of the core material.

In some embodiments, the material (eg, the material that forms the particle core) is at least about 20 degrees, at least about 30 degrees, at least about 40 degrees, at least about 50 degrees, at least about 60 degrees, at least about 70 degrees, about and a contact angle of at least 80 degrees, at least about 90 degrees, at least about 100 degrees, at least about 110 degrees, at least about 120 degrees, or at least about 130 degrees. In some embodiments, the material is about 160 degrees or less, about 150 degrees or less, about 140 degrees or less, about 130 degrees or less, about 120 degrees or less, about 110 degrees or less, about 100 degrees or less, about 90 degrees or less, about 80 degrees or less or less, or about 70 degrees or less. Combinations of the above-mentioned ranges are also possible (eg, a contact angle of greater than or equal to about 30 degrees and less than or equal to about 120 degrees). Other ranges are also possible.

Contact angle measurements can be made using a variety of techniques; Reference is made here to the measurement of the static contact angle between the beads of water and the pellets of the starting material which will be used to form the core. The material used to form the core was provided as a fine powder or otherwise ground into a fine powder using a mortar and pestle. To form the surface to measure against, the powder was packed using a 7 mm pellet die from International Crystal Labs. The material was added to the die and pressure was applied by hand to pack the powder into pellets, no pellet press or high pressure was used. The pellets were then suspended for testing so that the top and bottom portions of the pellets (surface water added and defined as opposite parallel surfaces each) did not contact any surface. This was done by not completely removing the pellets from the collar of the die set. The pellet thus touches the collar on the side and no contact on the top or the base at all. For contact angle measurement, water was added to the surface of the pellets over 30 seconds until beads of water with a steady contact angle were obtained. Water was added to the beads of water by submerging or contacting the tip of a pipette or syringe used for the addition of water to the beads. Once stable beads of water were obtained, images were taken and the contact angle was measured according to standard practice.

In embodiments where the core comprises an inorganic material (eg, for use as an imaging agent), the inorganic material is, for example, a metal (eg, Ag, Au, Pt, Fe, Cr, Co, Ni, Cu, Zn, and other transition metals), semiconductors (eg, silicon, silicon compounds and alloys, cadmium selenide, cadmium sulfide, indium arsenide, and indium phosphide), or insulators (eg, ceramics) , for example silicon oxide). The inorganic material may be present in any suitable amount, for example at least about 1 wt%, at least about 5 wt%, at least about 10 wt%, at least about 20 wt%, at least about 30 wt%, at least about 40 wt%, at least about 50 wt% % or more, about 75 wt% or more, about 90 wt% or more, or about 99 wt% or more, in the core. In one embodiment, the core is formed of 100 wt % inorganic material. In some cases, the inorganic material is about 100 wt% or less, about 90 wt% or less, about 80 wt% or less, about 70 wt% or less, about 60 wt% or less, about 50 wt% or less, about 40 wt% or less, about 30 wt% or less, about 20 wt% or less, about 10 wt% or less, about 5 wt% or less, about 2 wt% or less, or about 1 wt% or less. Combinations of the above-mentioned ranges are also possible (eg, present in an amount of at least about 1 wt % and up to about 20 wt %). Other ranges are also possible.

The core may, in some cases, be in the form of quantum dots, carbon nanotubes, carbon nanowires, or carbon nanorods. In some cases, the core comprises or is formed of a material that is not of biological origin.

In some embodiments, the core comprises one or more organic materials, such as synthetic polymers and/or natural polymers. Examples of synthetic polymers include non-degradable polymers such as polymethacrylate and degradable polymers such as polylactic acid, polyglycolic acid and copolymers thereof. Examples of natural polymers include hyaluronic acid, chitosan, and collagen. Other examples of polymers that may be suitable for portions of the core include those herein suitable for forming coatings on particles, as described below. The polymer may be present in any suitable amount, for example, about 100 wt% or less, about 90 wt% or less, about 80 wt% or less, about 70 wt% or less, about 60 wt% or less, about 50 wt% or less, about 40 wt% or less. or less, about 30 wt% or less, about 20 wt% or less, about 10 wt% or less, about 5 wt% or less, about 2 wt% or less, or about 1 wt% or less in the core. In some cases, the polymer is at least about 1 wt%, at least about 5 wt%, at least about 10 wt%, at least about 20 wt%, at least about 30 wt%, at least about 40 wt%, at least about 50 wt%, at least about 75 It may be present in the core in an amount of at least about wt%, at least about 90 wt%, or at least about 99 wt%. Combinations of the above-mentioned ranges are also possible (eg, present in an amount of at least about 1 wt % and up to about 20 wt %). Other ranges are also possible. In one set of embodiments, the core is formed substantially free of polymeric components.

The core may have any suitable shape and/or size. In some cases, the core may be substantially spherical, non-spherical, elliptical, rod-shaped, pyramidal, cube-like, disk-shaped, wire-like, or irregularly shaped. The core may be, for example, about 10 μm or less, about 5 μm or less, about 1 μm or less, about 800 nm or less, about 700 nm or less, about 500 nm or less, 400 nm or less, 300 nm or less, about 200 nm or less, about 100 nm or less, about 75 nm or less, about 50 nm or less, about 40 nm or less, about 35 nm or less, about 30 nm or less, about 25 nm or less, about 20 nm or less, about 15 nm or less, or about 5 nm or less It may have a maximum or minimum cross-sectional dimension. In some cases, the core is, for example, about 5 nm or more, about 20 nm or more, about 50 nm or more, about 100 nm or more, about 200 nm or more, about 300 nm or more, about 400 nm or more, about 500 nm or more, It may have a maximum or minimum cross-sectional dimension of at least about 1 μm, or at least about 5 μm. Combinations of the above-mentioned ranges are also possible (eg, a maximum or minimum cross-sectional dimension of about 50 nm or more and about 500 nm or less). Other ranges are also possible. In some embodiments, the sizes of the cores formed by the methods described herein have a Gaussian-type distribution. Unless otherwise specified, measurements of particle/core size herein refer to the smallest cross-sectional dimension.

Those skilled in the art are familiar with techniques for measuring the size (eg, minimum or maximum cross-sectional dimension) of a particle. Examples of suitable techniques include (DLS), transmission electron microscopy, scanning electron microscopy, electroresistance counting and laser diffraction. Other suitable techniques are known to those skilled in the art or skilled in the art. Although many methods for determining the size of a particle are known, the sizes described herein (eg, average particle size, thickness) refer to those measured by dynamic light scattering.

Methods for Forming Core Particles and Coated Particles

The core particles described herein may be formed by any suitable method. In some embodiments, a milling process may be used to reduce the size of the solid material to form particles in the micrometer to nanometer particle size range. Dry and wet milling processes such as jet milling, cryo-milling, ball milling, media milling, and homogenization are known and can be used with the methods described herein. Generally, in wet milling processes, a suspension of the material used as the core is mixed with the milling media with or without excipients to reduce the particle size. Dry milling is a process in which a material used as a core is mixed with a milling media with or without excipients to reduce particle size. In the cryo-milling process, a suspension of the material used as the core is mixed with the milling media with or without excipients under cooled temperature.

In some embodiments, the core particles described herein can be prepared by nanomilling a solid material (eg, a pharmaceutical) in the presence of one or more stabilizing/surface-altering agents. Small particles of solid material require the presence of one or more stabilizing/surface-altering agents, particularly on the surface of the particles, to stabilize the suspension of particles in liquid solution without agglomeration or aggregation. can do. In some such embodiments, the stabilizer may act as a surface-altering agent to form a coating on the particle.

As described herein, in some embodiments, methods of forming core particles include selecting suitable stabilizers for nanomilling and also to form a coating on the particles to render the particles mucus penetrating. For example, as described in more detail below, a well-established polymer-based MPP due to 200-500 nm nanoparticles of the model compound pyrene prepared by nanomilling pyrene in the presence of ^{Pluronic ® F127.} It has been demonstrated that results in particles capable of penetrating physiological mucus samples at the same rate as Interestingly, it was observed that only a few of the stabilizers/surface modifiers tested met the criteria for being suitable for nanomilling and also for forming coatings on particles, making them mucus penetrating, as described in more detail below. became

In a wet milling process, a dispersion (e.g., a dispersion containing one or more stabilizing agents (e.g., surface modifiers), a grinding medium, a solid being milled (e.g., a solid drug), and a solvent (e.g., Milling can be carried out in an aqueous dispersion. Any suitable amount of stabilizing/surface-altering agents may be included in the solvent. In some embodiments, the stabilizer/surface modifier is at least about 0.001% (wt% or %wt to volume (w:v)) of the solvent, at least about 0.01%, at least about 0.1%, at least about 0.5%, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 10% or more, about 12% or more, about 15% or more, about 20% or more, about 40% or more, about 60% or more, or about 80% or more. In some cases, the stabilizer may be present in the solvent in an amount of about 100% (eg, when the stabilizer/surface-altering agent is the solvent). In other embodiments, the stabilizer is about 100% or less, about 80% or less, about 60% or less, about 40% or less, about 20% or less, about 15% or less, about 12% or less, about 10% or less, It may be present in the solvent in an amount of about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less. Combinations of the aforementioned ranges are also possible (eg, in an amount of about 5% or less and about 1% or more of solvent). Other ranges are also possible. The specific range selected is a factor that can affect the ability of the particle to penetrate mucus, such as the stability of the coating of the stabilizer/surface-altering agent on the particle surface, the average thickness of the coating of the stabilizer/surface-altering agent on the particle, the particle size. The orientation of the stabilizer/surface modifier of the phase, the density of the stabilizer/surface modifier on the particle, the stabilizer:drug ratio, the drug concentration, the size and polydispersity of the particles formed, and the morphology of the particles formed.

The agent (or salt thereof) may be present in the solvent in any suitable amount. In some embodiments, the agent (or salt thereof) is at least about 0.001% (wt% or %wt to volume (w:v)) of the solvent, at least about 0.01%, at least about 0.1%, at least about 0.5%, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 10% or more, about 12% or more, about present in the solvent in an amount of at least 15%, at least about 20%, at least about 40%, at least about 60%, or at least about 80%. In some cases, the agent (or salt thereof) is about 100% or less, about 90% or less, about 80% or less, about 60% or less, about 40% or less, about 20% or less, about 15% or less, about 12% or less of the solvent. % or less, about 10% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less may be present in the solvent. Combinations of the above-mentioned ranges are also possible (eg, in an amount of about 20% or less of about 1% or more of solvent). In some embodiments, the agent is present in the above ranges but w:v.

The ratio of stabilizer/surface-altering agent to agent (or salt thereof) in the solvent may also vary. In some embodiments, the ratio of stabilizer/surface-altering agent to drug (or salt thereof) is 0.001:1 or greater (weight ratio, molar ratio, or w:v ratio), 0.01:1 or greater, 0.01:1 or greater, 1: 1 or more, 2:1 or more, 3:1 or more, 5:1 or more, 10:1 or more, 25:1 or more, 50:1 or more, 100:1 or more, or 500:1 or more. In some cases, the ratio of stabilizer/surface-altering agent to drug (or salt thereof) is 1000:1 or less (weight ratio or molar ratio), 500:1 or less, 100:1 or less, 75:1 or less, 50:1 or less. , 25:1 or less, 10:1 or less, 5:1 or less, 3:1 or less, 2:1 or less, 1:1 or less, or 0.1:1 or less. Combinations of the above-mentioned ranges are possible (eg, 5:1 or greater and 50:1 or less). Other ranges are also possible.

Stabilizers/surface modifiers may be, for example, polymers or surfactants. Examples of polymers are those suitable for use in coatings as described in more detail below. Non-limiting examples of surfactants include L-α-phosphatidylcholine (PC), 1,2-dipalmitoylphosphatidylcholine (DPPC), oleic acid, sorbitan trioleate, sorbitan monooleate, sorbitan monolaurate, polyoxy ethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, natural lecithin, oleyl polyoxyethylene ether, stearyl polyoxyethylene ether, lauryl polyoxyethylene ether, block copolymers of oxyethylene and oxypropylene; Synthetic lecithin, diethylene glycol dioleate, tetrahydrofurfuryl oleate, ethyl oleate, isopropyl myristate, glyceryl monooleate, glyceryl monostearate, glyceryl monoricinolate, cetyl alcohol, stearyl alcohol, polyethylene glycol 400, cetyl pyridinium chloride, benzalkonium chloride, olive oil, glyceryl monolaurate, corn oil, cottonseed oil, and sunflower seed oil. Derivatives of the abovementioned compounds are also possible. Combinations of the above-mentioned compounds with others described herein may also be used as surface-altering agents in the particles of the present invention. As described herein, in some embodiments surface-altering agents may act as stabilizers, surfactants, and/or emulsifiers. In some embodiments, surface-altering agents can aid in particle transport in mucus.

In some embodiments, the stabilizing agent used for milling forms a coating on the particle surface, which coating renders the particle mucus penetrating, while in other embodiments, the stabilizing agent forms a coating on one or more other surfaces after the particle is formed. It should be noted that modifiers can be substituted. For example, in one set of methods, the first stabilizer/surface-altering agent may be used during the milling process and coat the surface of the core particle, followed by removing all or a portion of the first stabilizer/surface-altering agent. 2 Can be replaced with a stabilizer/surface modifier to coat all or part of the surface of the core particle. In some cases, the second stabilizer/surface-altering agent may render the particle more mucus penetrating than the first stabilizer/surface-altering agent. In some embodiments, it is possible to form a core particle having a coating comprising multiple surface-altering agents.

Any suitable grinding media may be used for milling. In some embodiments, ceramic and/or polymeric materials and/or metals may be used. Examples of suitable materials may include zirconium oxide, silicon carbide, silicon oxide, silicon nitride, zirconium silicate, yttrium oxide, glass, alumina, alpha-alumina, aluminum oxide, polystyrene, poly(methyl methacrylate), titanium, steel. have. The grinding media may have any suitable size. For example, the grinding media can have an average diameter of at least about 0.1 mm, at least about 0.2 mm, at least about 0.5 mm, at least about 0.8 mm, at least about 1 mm, at least about 2 mm, or at least about 5 mm. In some cases, the grinding media may have an average diameter of about 5 mm or less, about 2 mm or less, about 1 mm or less, about 0.8 mm or less, about 0.5 mm or less, or about 0.2 mm or less. Combinations of the above-mentioned ranges are also possible (eg, an average diameter of greater than or equal to about 0.5 millimeters and less than or equal to about 1 mm). Other ranges are also possible.

Any suitable solvent may be used for milling. The choice of solvent will depend, among other factors, on the solid material being milled (eg, pharmaceutical), the particular type of stabilizing/surface-altering agent used (eg, capable of rendering the particles mucus penetrating); This may be determined by factors such as the grinding material used. Suitable solvents may be those that do not substantially dissolve the solid or ground material, but do solubilize the stabilizer/surface-altering agent to a suitable extent. Non-limiting examples of solvents include other ingredients such as pharmaceutical excipients, polymers, agents, salts, preservatives, viscosity modifiers, tonicity modifiers, taste masking agents, antioxidants, pH adjusting agents, and water, buffer solutions, other aqueous solutions, alcohols (eg, ethanol, methanol, butanol), and mixtures thereof, which may optionally include other pharmaceutical excipients. In other embodiments, organic solvents may be used. The medicament may have a suitable solubility in any of these or other solvents, such as solubility in one or more of the ranges described above for solubility in water or solubility in coating solutions.

In other embodiments, the core particles may be formed by precipitation techniques. A precipitation technique (eg, microprecipitation technique, nanoprecipitation technique) can include forming a first solution comprising a substance (eg, a pharmaceutical) used as a core and a solvent, wherein the substance is in the solvent practically available. The solution may be added to a second solution comprising another solvent, wherein the material is substantially insoluble, thereby forming a plurality of particles comprising the material. In some cases, one or more surface-altering agents, surfactants, substances, and/or bioactive agents may be present in the first and/or second solution. A coating may form during the process of precipitating the core (eg, the precipitation and coating steps may be performed substantially simultaneously). In another embodiment, the particles are first formed using precipitation techniques, and then the particles are coated with a surface-altering agent.

In some embodiments, precipitation techniques may be used to form particles (eg, nanocrystals) of a salt of a drug. In general, precipitation techniques involve dissolving the material used as the core in a solvent and then adding it to a miscible anti-solvent with or without excipients to form the core particles. This technique may be useful for preparing particles of a pharmaceutical agent (eg, an agent having a relatively high water solubility) that is soluble in aqueous solution. In some embodiments, an agent having one or more charged or ionizable groups can interact with a counter ion (eg, a cation or anion) to form a salt complex. For example, the drug tenofovir (TFV) interacts very strongly with zinc cations via phosphonate groups and purine ring structures. This interaction with zinc results in TFV precipitation into crystals that can be stabilized by the coatings described herein to stop agglomeration.

A variety of counter ions can be used to form salt complexes, including metals (eg, alkali metals, alkaline earth metals and transition metals). Non-limiting examples of cationic counter ions include zinc, calcium, aluminum, zinc, barium, magnesium, and copper. Non-limiting examples of anionic counter ions include phosphates, carbonates, and fatty acids. The counter ion may be, for example, monovalent, divalent, or trivalent. Other counter ions are known in the art and may be used in the embodiments described herein.

A variety of different acids can be used in the precipitation process. In some embodiments, suitable acids may include decanoic acid, hexanoic acid, mucinic acid, octanoic acid. In other embodiments, suitable acids are acetic acid, adipic acid, L-ascorbic acid, L-aspartic acid, capric acid (decanoic acid), carbonic acid, citric acid, fumaric acid, galactaric acid, D-glucoheptonic acid, D-glu Conic acid, D-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrochloric acid, DL-lactic acid, lauric acid, maleic acid, (-)-L-malic acid, palmitate acid, phosphoric acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, (+)-L-tartaric acid, or thiocyanate. In other embodiments, suitable acids are alginic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, caprylic acid (octanoic acid), cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, Ethanesulfonic acid, 2-hydroxy-, gentisic acid, glutaric acid, 2-oxo-, isobutyric acid, lactobionic acid, malonic acid, methanesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 2-naphthoic acid, 1-hydroxy-, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmic acid, (embonic acid), propionic acid, (-)-L-pyroglutamic acid, or p -toluenesulfonic acid. In another embodiment suitable acids are acetic acid, 2,2-dichloro-, benzoic acid, 4-acetamido-, (+)-camphor-10-sulfonic acid, caproic acid (hexanoic acid), cinnamic acid, formic acid, bromination hydrochloric acid, DL-mandelic acid, nitric acid, salicylic acid, salicylic acid, 4-amino-, or undecylenic acid (undec-10-enoic acid). Mixtures of one or more of these acids may also be used.

A variety of different bases can be used in the precipitation process. In some embodiments, suitable bases include ammonia, L-arginine, calcium hydroxide, choline, glucamine, N -methyl-, lysine, magnesium hydroxide, potassium hydroxide, or sodium hydroxide. In other embodiments, suitable bases are benetamine, benzathine, betaine, theanol, diethylamine, ethanol, 2-(diethylamino)-, hydrabamine, morpholine, 4-(2-hydroxyethyl )-, pyrrolidine, 1-(2-hydroxyethyl)-, or tromethamine. In other embodiments, suitable bases are diethanolamine (2,2'-iminobis(ethanol)), ethanolamine (2-aminoethanol), ethylenediamine, 1 H -imidazole, piperazine, triethanolamine (2, 2',2"-nitrilotris (ethanol)), or zinc hydroxide. Mixtures of one or more of these bases may also be used.

Any suitable solvent can be used for precipitation, including the solvents described herein that can be used for milling. In one set of embodiments, aqueous solutions (eg, water, buffer solutions, other aqueous solutions, alcohols (eg, ethanol, methanol, butanol), which may optionally include other ingredients such as pharmaceutical excipients, polymers, and pharmaceuticals) ), and mixtures thereof).

In the precipitation process, the salt may have a lower water solubility (or solubility in the solvent containing the salt) than the drug in its non-salt form. The water solubility (or solubility in solvent) of the salt is, for example, about 5 mg/mL or less, about 2 mg/mL or less, about 1 mg/mL or less, about 0.5 mg/mL or less, about 0.1 mg/mL or less at 25°C. or less, about 0.05 mg/mL or less, about 0.01 mg/mL or less, about 1 μg/mL or less, about 0.1 μg/mL or less, about 0.01 μg/mL or less, about 1 ng/mL or less, about 0.1 ng/mL or less, or about 0.01 ng/mL or less. In some embodiments, the salt is at least about 1 pg/mL, at least about 10 pg/mL, at least about 0.1 ng/mL, at least about 1 ng/mL, at least about 10 ng/mL, at least about 0.1 μg/mL, about 1 μg/mL or more, about 5 μg/mL or more, about 0.01 mg/mL or more, about 0.05 mg/mL or more, about 0.1 mg/mL or more, about 0.5 mg/mL or more, about 1.0 mg/mL or more, about 2 It may have a solubility in water (or solubility in solvent) of mg/mL. Combinations of the above-mentioned ranges are possible (eg, solubility in water (or solubility in solvent) of at least about 0.001 mg/mL and less than or equal to about 1 mg/mL). Other ranges are also possible. Salts may have aqueous solubility in these or other ranges at any point across the pH range (eg, pH 1 to pH 14).

In some embodiments, the solvent used for precipitation comprises one or more surface-altering agents as described herein, and as it precipitates from solution, a coating of the one or more surface-altering agents may form around the particles. The surface-altering agent is at any suitable concentration, such as at least about 0.001 (w/v), at least about 0.005% (w/v), at least about 0.01% (w/v), at least about 0.05% (w/v) in aqueous solution. , at least about 0.1% (w/v), at least about 0.5% (w/v), at least about 1% (w/v), or at least about 5% (w/v) in the solvent. In some cases, the surface-altering agent is about 5% (w/v) or less, about 1% (w/v) or less, about 0.5% (w/v) or less, about 0.1% (w/v) or less, about 0.05 % (w/v) or less, about 0.01% (w/v) or less, or about 0.005% (w/v) or less in the solvent. Combinations of the above-mentioned ranges are also possible (eg, a concentration of greater than or equal to about 0.01% (w/v) and less than or equal to about 1% (w/v)). Other ranges are also possible.

Another exemplary method of forming the core particles includes freeze-drying techniques. In this technique, the agent or salt thereof may be dissolved in an aqueous solution, optionally containing a surface-altering agent. Counter ions can be added to the solution, and the solution can be flash frozen (quick-freeze) and freeze-dried immediately. The dry powder can be reconstituted in a suitable solvent (eg, an aqueous solution, such as water) at the desired concentration.

The counter ion may be added to the solvent for lyophilization to any suitable extent. In some cases, the ratio of counter ion to agent (eg, salt) is 0.1:1 (weight ratio or molar ratio) or greater, 1:1 or greater, 2:1 or greater, 3:1 or greater, 5:1 or greater, 10 1:1 or greater, 25:1 or greater, 50:1 or greater, or 100:1 or greater. In some cases, the ratio of counter ion to agent (eg, salt) is 100:1 (weight ratio or molar ratio) or less, 75:1 or less, 50:1 or less, 25:1 or less, 10:1 or less, 5 :1 or less, 3:1 or less, 2:1 or less, 1:1 or less, or 0.1:1 or less. Combinations of the above-mentioned ranges are possible (eg, a ratio of at least 5:1 and up to 50:1). Other ranges are also possible.

When the surface-altering agent is present in the solvent prior to lyophilization, it may be at any suitable concentration, such as at least about 0.001% (w/v), at least about 0.005% (w/v), at least about 0.01% (w/v) in aqueous solution. , at least about 0.05% (w/v), at least about 0.1% (w/v), at least about 0.5% (w/v), at least about 1% (w/v), or at least about 5% (w/v) It may be present in higher concentrations. In some cases, the surface-altering agent is about 5% (w/v) or less, about 1% (w/v) or less, about 0.5% (w/v) or less, about 0.1% (w/v) or less, about 0.05 % (w/v) or less, about 0.01% (w/v) or less, or about 0.005% (w/v) or less in the solvent. Combinations of the above-mentioned ranges are also possible (eg, a concentration of greater than or equal to about 0.01% (w/v) and less than or equal to about 1% (w/v)). Other ranges are also possible.

The concentration of the surface-altering agent present in the solvent may be above or below the critical micelle concentration (CMC) of the surface-altering agent, depending on the particular surface-altering agent used. For example, as described in the Examples section, F127 concentrations of both above (1%) and below (0.08%) CMC of F127 can be used to coat stable nanocrystalline particles of the drug tenofovir. However, the nanocrystals of acyclovir monophosphate were much more sensitive to the surfactant concentration, and stable nanocrystal particles could only be formed when using a F127 concentration below the CMC (~0.1%).

In other embodiments, stable particles can be formed by adding an excess of counter ion to a solution containing the agent. The precipitate can then be washed by various methods such as centrifugation. The resulting slurry may be sonicated. One or more surface-altering agents may be added to stabilize the resulting particles.

Other methods of forming particles of the medicament are also possible. So-called top-down techniques include, for example, milling techniques and high-pressure homogenization. In high pressure homogenization, the suspension of the material used as the core is forced under pressure through a gap, valve, or aperture to reduce the particle size. So-called bottom-up techniques include, for example, precipitation, emulsification, wherein a substance used as a core dissolved in a solvent is added to an immiscible anti-solvent with or without excipients to form core particles; and spray drying, wherein a solution of the material used as the core is sprayed into a gaseous anti-solvent to form the core particles.

Combinations of the methods described herein with other methods are also possible. For example, in some embodiments, the core of the drug is first formed by precipitation, and then the size of the core is further reduced by a milling process.

After particle formation of the medicament, the particles may optionally be exposed to a solution comprising a (second) surface-altering agent capable of associating with and/or coating the particles. In embodiments where the medicament already comprises a coating of the first surface-altering agent, all or a portion of the second surface-altering agent may be replaced with the second stabilizer/surface-altering agent to coat all or a portion of the particle surface. In some cases, the second surface-altering agent may render the particle more mucus penetrating than the first surface-altering agent. In other embodiments, particles having a coating comprising multiple surface-altering agents may be formed (eg, in a single layer or in multiple layers). In other embodiments, it is possible to form particles having multiple coatings (eg, each coating optionally comprising a different surface-altering agent). In some cases, the coating is in the form of a single layer of a surface-altering agent. Other configurations are also possible.

In any one of the methods described herein, at least about 1 minute, at least about 2 minutes, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 30 minutes, at least about 60 minutes, The particles may be coated with the surface-altering agent by incubating the particles in solution with the surface-altering agent for an or longer period of time. In some cases, the incubation may be conducted for a period of about 10 hours or less, about 5 hours or less, or about 60 minutes or less. Combinations of the above-mentioned ranges are also possible (eg, an incubation period of about 2 minutes or more of 60 minutes or less).

particle coating

As shown in the embodiment illustrated in FIG. 1 , the core 16 may be surrounded by a coating 20 comprising one or more surface-altering agents. In some embodiments, the coating is formed of one or more surface-altering agents or other molecules disposed on the surface of the core. The specific chemical makeup and/or components of the coating and surface modifying agent(s) may be selected to impart specific functionality to the particle, such as enhanced transport across the mucosal barrier.

It should be understood that the coating surrounding the core need not completely surround the core, although such an embodiment may be possible. For example, the coating may comprise at least about 10%, at least about 30%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99% of the surface area of the core. It can enclose more than In some cases, the coating substantially surrounds the core. In other cases, the coating completely surrounds the core. In other embodiments, the coating encloses about 100% or less, about 90% or less, about 80% or less, about 70% or less, about 60% or less, or about 50% or less of the surface area of the core. Combinations of the above-mentioned ranges are also possible (eg encompassing more than 80% and less than 100% of the surface area of the core).

The components of the coating may be distributed evenly over the surface of the core in some cases, and unevenly in other cases. For example, the coating may in some cases include portions (eg, hollow) that do not contain any material. If desired, the coating may be designed to allow penetration and/or transport of certain molecules and components into or out of the coating, but may prevent penetration and/or transport of other molecules and components into or out of the coating. have. The ability of a particular molecule to penetrate and/or transport into and/or across a coating depends, for example, on the packing density of the surface-altering agent forming the coating and the chemistry and It may vary depending on physical properties. As described herein, the coating may in some embodiments comprise one layer of material, or multiple layers of material. There may be a single type of surface-altering agent, or multiple forms of the surface-altering agent.

The coating of particles may have any suitable thickness. For example, the coating may be at least about 1 nm, at least about 5 nm, at least about 10 nm, at least about 30 nm, at least about 50 nm, at least about 100 nm, at least about 200 nm, at least about 500 nm, at least about 1 μm. or more, or an average thickness of about 5 μm or more. In some cases, the average thickness of the coating is about 5 μm or less, about 1 μm or less, about 500 nm or less, about 200 nm or less, about 100 nm or less, about 50 nm or less, about 30 nm or less, about 10 nm or less, or about 5 nm or less. Combinations of the above-mentioned ranges are also possible (eg, an average thickness of about 1 nm to about 100 nm or less). Other ranges are also possible. For particles with multiple coatings, each coating layer can have one of the thicknesses described above.

In some embodiments, the compositions and methods described herein may enable coating of a core particle with a hydrophilic surface-altering moiety without the need for covalent linkage of the surface-altering moiety to the core surface. In some such embodiments, a core having a hydrophobic surface may be coated with the polymers described herein, thereby resulting in a number of surface modifying moieties on the core surface without substantially altering the characteristics of the core itself. For example, the surface-altering agent can be adsorbed to the outer surface of the core particle. However, in other embodiments, the surface-altering agent is covalently linked to the core particle.

In certain embodiments where the surface-altering agent is adsorbed onto the surface of the core, the surface-altering agent can be in equilibrium with other molecules of the surface-altering agent, optionally with other components (eg, in the composition/formulation), and in solution. In some cases, the adsorbed surface-altering agent may be present on the surface of the core at the densities described herein. The density can be an average density because the surface modifier is in equilibrium with the other components in solution.

The coatings and/or surface-altering agents of the particles described herein may include any suitable material, such as a hydrophobic material, a hydrophilic material, and/or an amphiphilic material. In some embodiments, the coating comprises a polymer. In certain embodiments, the polymer is a synthetic polymer (ie, a polymer that is not naturally produced). In other embodiments, the polymer is a natural polymer (eg, protein, polysaccharide, rubber). In certain embodiments, the polymer is a surface active polymer. In certain embodiments, the polymer is a non-ionic polymer. In certain embodiments, the polymer is a nonionic block copolymer. In some embodiments, the polymer can be, for example, a diblock copolymer, a triblock copolymer, for example, in which one block is a hydrophobic polymer and another block is a hydrophilic polymer. Polymers may be charged or uncharged.

In some embodiments, the particles described herein comprise a coating comprising a block copolymer having relatively hydrophilic blocks and relatively hydrophobic blocks. In some cases, the hydrophilic block may be substantially present on the outer surface of the particle. For example, the hydrophilic block can form the majority of the outer surface of the coating and can help stabilize the particles in the aqueous solution containing the particles. The hydrophobic block may be substantially present on the interior of the coating and/or on the surface of the core particle, for example, to facilitate adhesion of the coating to the core. In some cases, the coating comprises a surface modifier comprising a triblock copolymer, wherein the triblock copolymer comprises a hydrophilic block-hydrophobic block-hydrophilic block configuration.

The molecular weights of the hydrophilic and hydrophobic blocks of the triblock copolymer may be selected to reduce mucoadhesion of the core and ensure sufficient association of the triblock copolymer with the core, respectively. As described herein, the molecular weight of the hydrophobic block of the triblock copolymer can be selected to increase the likelihood that proper association of the triblock copolymer with the core will occur, thereby causing the triblock copolymer to remain attached to the core. have. Surprisingly, in certain embodiments, too low molecular weight (eg, about 2 kDa or less) of the hydrophobic block of the triblock copolymer does not allow sufficient adhesion with the hydrophobic core and the triblock copolymer, and thus It has been found that particles with hydrophobic blocks cannot exhibit sufficient reduced mucoadhesion.

In certain embodiments, the molecular weight of the hydrophobic block of the triblock copolymer (e.g., the PPO block of the triblock copolymer PEG-PPO-PEG, wherein the PEG block may be interchanged with the PEO block) has a molecular weight of about 2 or more, about 3 kDa or more, about 4 kDa or more, about 5 kDa or more, about 6 kDa or more, about 10 kDa or more, about 20 kDa or more, or about 50 kDa or more. In some embodiments, the molecular weight of the hydrophobic block is about 100 kDa or less, about 80 kDa or less, about 50 kDa or less, about 20 kDa or less, about 15 kDa or less, about 13 kDa or less, about 12 kDa or less, about 10 kDa or less, about 8 kDa or less, or about 6 kDa or less. Combinations of the aforementioned ranges are also possible (eg, greater than or equal to about 3 kDa and less than or equal to about 15 kDa). Other ranges are also possible.

It has also been found that, in certain embodiments, a sufficient amount of hydrophilic block (as a function of the total weight of the polymer) is required for the particles to exhibit sufficient reduced mucoadhesion. For example, in certain embodiments, the hydrophilic blocks comprising at least about 15 wt% (e.g., at least about 20 wt%, at least about 25 wt%, at least about 30 wt%) of the triblock copolymer comprise particles. Mucoadhesion was generally observed with particles having a weight percentage of hydrophilic block below this limit, while allowing mucus penetration. In some embodiments, the hydrophilic block of the triblock copolymer comprises at least about 15 wt%, at least about 20 wt%, at least about 25 wt%, at least about 30 wt%, at least about 35 wt%, at least about 40 wt% of the triblock copolymer. at least about 45 wt%, at least about 50 wt%, at least about 55 wt%, at least about 60 wt%, at least about 65 wt%, or at least about 70 wt%. In some embodiments, the hydrophilic blocks of the triblock copolymer make up about 90 wt% or less, about 80 wt% or less, about 60 wt% or less, about 50 wt% or less, or about 40 wt% or less of the triblock copolymer. do. Combinations of the above-mentioned ranges are also possible (eg, at least about 30 wt % and up to about 80 wt %). Other ranges are also possible.

In some embodiments, the molecular weight of the hydrophilic block (e.g., the PEG (or PEO) block of the triblock copolymer PEG-PPO-PEG, wherein the PEG block may be interchanged with the PEO block) is at least about 0.05 kDa , about 0.1 kDa or more, about 0.2 kDa or more, about 0.3 kDa or more, about 0.4 kDa or more, about 0.5 kDa or more, about 1 kDa or more, about 2 kDa or more, about 3 kDa or more, about 4 kDa or more, about 5 kDa or more , about 6 kDa or more, about 8 kDa or more, about 10 kDa or more, about 20 kDa or more, or about 50 kDa or more.In certain embodiments, the molecular weight of the hydrophilic block is about 100 kDa or less, about 80 kDa or less, about 50 kDa or less, about 20 kDa or less, about 15 kDa or less, about 10 kDa or less, about 9 kDa or less, about 8 kDa or less, about 7 kDa or less, about 6 kDa or less, about 5 kDa or less, about 3 kDa or less, about 2 kDa or less, or about 1 kDa or less.Combination of the above-mentioned ranges is also possible (for example, from about 0.1 kDa to about 3 kDa).Other ranges are also possible.Two hydrophilic blocks are hydrophobic blocks. In embodiments on the aspect of the two hydrophilic blocks, the molecular weights may be substantially the same or different.

In certain embodiments, the polymer of the surface modifier comprises a polyether moiety. In certain embodiments, the polymer comprises a polyalkylether moiety. In certain embodiments, the polymer comprises a polyethylene glycol tail. In certain embodiments, the polymer comprises a polypropylene glycol core. In certain embodiments, the polymer comprises polybutylene glycol as the core. In certain embodiments, the polymer comprises polypentylene glycol as the core. In certain embodiments, the polymer comprises polyhexylene glycol as the core. In certain embodiments, the polymer is a triblock copolymer of one of the polymers described herein. As disclosed herein, any description of PEG may be replaced by polyethylene oxide (PEO), and any description of PEO may be replaced by PEG.

In certain embodiments, the polymer is a triblock copolymer of a polyalkyl ether (eg, polyethylene glycol, polypropylene glycol) and another polymer. In certain embodiments, the polymer is a triblock copolymer of a polyalkyl ether and another polyalkyl ether. In certain embodiments, the polymer is a triblock copolymer of polyethylene glycol and another polyalkyl ether. In certain embodiments, the polymer is a triblock copolymer of polypropylene glycol and another polyalkyl ether. In certain embodiments, the polymer is a triblock copolymer having one or more units of polyalkyl ethers. In certain embodiments, the polymer is a triblock copolymer of two different polyalkyl ethers. In certain embodiments, the polymer is a triblock copolymer comprising polyethylene glycol units. In certain embodiments, the polymer is a triblock copolymer comprising polypropylene glycol units. In certain embodiments, the polymer is a triblock copolymer of more hydrophobic units flanked by two or more hydrophilic units. In certain embodiments, the hydrophilic units are of the same type of polymer. In certain embodiments, the polymer comprises polypropylene glycol units flanked by two or more hydrophilic units. In certain embodiments, the polymer comprises two polyethylene glycol units flanked by more hydrophobic units. In certain embodiments, the polymer is a triblock copolymer having polypropylene glycol units flanked by two polyethylene glycol units. The molecular weights of the two blocks flanking the central block may be substantially the same or different.

In certain embodiments, the polymer is of the formula:

where n is all integers from 2 to 1140; m is any integer from 2 to 1730. In certain embodiments, n is all integers from 10 to 170. In certain embodiments, m is all integers from 5 to 70. In certain embodiments, n is at least 2 times m, at least 3 times m, or at least 4 times m.

In certain embodiments, it comprises a (poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol)) triblock copolymer (hereinafter “PEG-PPO-PEG triblock copolymer”). and surface-altering agents. As described herein, PEG blocks may be interchanged with PEO blocks in some embodiments. The molecular weights of the PEG (or PEO) and PPO segments of the PEG-PPO-PEG triblock copolymer can be selected to reduce adhesion of the particles, as described herein. While not wishing to be bound by theory, particles having a coating comprising a PEG-PPO-PEG triblock copolymer were controlled, at least in part, due to the display of multiple PEG (or PEO) segments on the particle surface. It may have reduced mucoadhesion compared to the particles. The PPO segment can be attached to the core surface (eg, when the core surface is hydrophobic), thus allowing for a strong association between the core and the polymer. In some cases, the PEG-PPO-PEG triblock copolymer is associated with the core through non-covalent interactions. For comparison purposes, the control particle may be, for example, a carboxylate-modified polystyrene particle of a similar size to the corresponding coated particle.

In certain embodiments, the surface-altering agent comprises a polymer comprising a poloxamer having the ^{trade name Pluronic ® . Pluronic ®} polymers that may be useful in the embodiments described herein include F127, F38, F108, F68, F77, F87, F88, F98, L101, L121, L31, L35, L43, L44, L61, L62, L64, L81, L92, N3, P103, P104, P105, P123, P65, P84, and P85.

Examples of molecular weights of specific Pluronic ^® molecules are shown in Table 1.

Although other ranges are possible and useful in certain embodiments described herein, in some embodiments, the hydrophobic block of the PEG-PPO-PEG triblock copolymer has one of the molecular weights described above (e.g., about 3 kDa or more, about 15 kDa or less), the combined hydrophilic blocks have a weight percentage relative to the polymer in one of the ranges described above (e.g., about 15 wt % or more, about 20 wt % or more, about 25 wt % or more, or greater than or equal to about 30 wt %, and less than or equal to about 80 wt %). ^{Specific Pluronic ®} polymers that fall within these criteria include, for example, F127, F108, P105 and P103. Surprisingly, and as described in more detail in the Examples, it has been found that these specific Pluronic ^® polymers render the particles more mucus penetrating than ^{other Pluronic ®} polymers tested that do not fall under these criteria. In addition, other agents that do not render the particles mucus penetrating include certain polymers such as polyvinylpyrrolidone (PVP/Kollidon), polyvinyl alcohol-polyethylene glycol graft-copolymer (Kollicoat IR) , hydroxypropyl methylcellulose (Methocel); oligomers such as Tween 20, Tween 80 such as solutol HS 15, Triton X100, tyloxapol, Cremophor RH 40; small molecules such as Span 20, Span 80, octyl glucoside, cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS).

Although much of the description herein relates to coatings comprising a hydrophilic block-hydrophobic block-hydrophilic block configuration (eg, PEG-PPO-PEG triblock copolymer), coatings are limited to these configurations and materials. However, it should be understood that other configurations and materials are possible. For example, a particle may include more than one coating (eg, at least two, three, four, five, or more coatings), each coating being mucus penetrating It need not be formed of or contain a substance. In some cases, the intermediate coating (ie, the coating between the core surface and the outer coating) may include a polymer that promotes adhesion of the outer coating to the core surface. In many embodiments, the outer coating of the particle comprises a polymer comprising a material that facilitates transport of the particle through mucus.

As such, the coating (eg, inner coating, intermediate coating, and/or outer coating) may include any suitable polymer. In some cases, the polymer may be biocompatible and/or biodegradable. In some cases, the polymeric material may include more than one type of polymer (eg, at least two, three, four, five, or more polymers). In some cases, the polymer may be a random copolymer or a block copolymer (eg, a diblock copolymer, a triblock copolymer) as described herein.

Non-limiting examples of suitable polymers include polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA) , poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) ( PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO) -co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylate, polyurethane, poly-L-lysine ( PLL), hydroxypropyl methacrylate (HPMA), poly(ethylene glycol), poly-L-glutamic acid, poly(hydroxy acid), polyanhydride, polyorthoester, poly(ester amide), polyamide, polyester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxide (PEO), polyalkylene terephthalates such as poly (ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ether, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxane, Polystyrene (PS), polyurethane, derivatized cellulose such as alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitro cellulose, hydroxypropylcellulose, carboxymethylcellulose, polymers of acrylic acid such as poly(methyl(meth) acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly( Isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly( isobutyl acrylate), poly(octadecyl acrylate) (collectively referred to herein as “polyacrylic acid”), and copolymers and mixtures thereof, polydioxane and copolymers thereof, polyhydroxyalkanoates, poly propylene fumarate), polyoxymethylene, poloxamer, poly(ortho)ester, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), and trimethylene carbonate, polyvinylpi contains rolidone.

The molecular weight of the polymer may be different. In some embodiments, the molecular weight is at least about 0.5 kDa, at least about 1 kDa, at least about 2 kDa, at least about 3 kDa, at least about 4 kDa, at least about 5 kDa, at least about 6 kDa, at least about 8 kDa, at least about 10 kDa , about 12 kDa or more, about 15 kDa or more, about 20 kDa or more, about 30 kDa or more, about 40 kDa or more, or about 50 kDa or more. In some embodiments, the molecular weight is about 50 kDa or less, about 40 kDa or less, about 30 kDa or less, about 20 kDa or less, about 12 kDa or less, about 10 kDa or less, about 8 kDa or less, about 6 kDa or less, about 5 kDa or less. or less, or about 4 kDa or less. Combinations of the above-mentioned ranges are possible (eg, molecular weight of about 2 kDa or more and about 15 kDa or less). Other ranges are also possible. Molecular weight can be determined using any known technique, such as light-scattering and gel permeation chromatography. Other methods are known in the art.

In certain embodiments, the polymer is biocompatible, ie, the polymer typically does not induce an adverse response upon insertion or injection into a living body; For example, it does not involve significant inflammatory and/or acute rejection of polymers by the immune system, for example through T-cell-mediated responses. Of course, it will be appreciated that "biocompatibility" is a relative term, and that some degree of immune response is expected for polymers that are even highly compatible with living tissues. However, as used herein, “biocompatible” refers to an acute rejection of a substance by at least a portion of the immune system, i.e., a non-biocompatible substance implanted in a subject elicits an immune response in the subject, which causes the immune system to The rejection of the substance by One simple test to determine biocompatibility is to expose the polymer to cells in vitro; Biocompatible polymers are polymers that typically do not cause much cell death at moderate concentrations, for example at concentrations of about 50 micrograms/10 ^{6 cells.} For example, biocompatible polymers can induce up to about 20% cell death upon exposure to cells such as fibroblasts or epithelial cells, even if not phagocytosed or otherwise taken up by such cells. In some embodiments, a substance is "biocompatible" if its addition to cells in vitro results in 20% or less cell death and its administration in vivo does not induce unwanted inflammation or other such side effects.

In certain embodiments, a biocompatible polymer may be biodegradable, i.e., the polymer is chemically and/or biologically (e.g., cellular machinery ) or by hydrolysis). For example, the polymer may be one that hydrolyzes spontaneously upon exposure to water (eg, in a subject) and/or the polymer will degrade upon exposure to heat (eg, at a temperature of about 37° C.) can Degradation of the polymer can occur at various rates, depending on the polymer or copolymer used. For example, the half-life of a polymer (the time at which 50% of the polymer is degraded to monomeric and/or other non-polymeric moieties) can be on the order of days, weeks, months, or years, depending on the polymer. Polymers may, in some cases, be biologically degraded, eg, by enzymatic activity or cellular machinery, eg, through exposure to lysozyme (eg, having a relatively low pH). In some cases, the polymer is a monomeric and/or other non-polymeric moiety that the cells can reuse or process without significant toxic effects on the cells (i.e., less than about 20% of the cells die when the component is added to the cells in vitro). It can be broken down into tea. For example, polylactide may be hydrolyzed to form lactic acid, polyglycolide may be hydrolyzed to form glycolic acid, and the like.

Examples of biodegradable polymers include poly(ethylene glycol)-poly(propylene oxide)-poly(ethylene glycol) triblock copolymer, poly(lactide) (or poly(lactic acid)), poly(glycolide) (or poly (glycolic acid)), poly(orthoester), poly(caprolactone), polylysine, poly(ethylene imine), poly(acrylic acid), poly(urethane), poly(anhydride), polyester), poly(trimethylene) carbonate), poly(ethyleneimine), poly(acrylic acid), poly(urethane), poly(beta amino ester), etc., and copolymers or derivatives of these and/or other polymers, such as poly(lactide- co-glycolide) (PLGA).

In certain embodiments, the polymer is capable of biodegradation within a time period acceptable for the desired application. In certain embodiments, such as in in vivo therapy, such degradation usually occurs at about 5 years, 1 year, 6 months, 3 months, 1 month, on exposure to a physiological solution having a pH of 6-8 at a temperature of 25-37°C; 15 days, 5 days, 3 days or less, or even 1 day or less (eg, 1-4 hours, 4-8 hours, 4-24 hours, 1-24 hours). In other embodiments, the polymer degrades in a period of from about 1 hour to several weeks, depending on the desired application.

Although the coatings and particles described herein can comprise polymers, in some embodiments, the particles described herein comprise a hydrophobic material that is not a polymer (eg, non-polymer) and is not a pharmaceutical. For example, all or a portion of the particles may be coated with a passivation layer in some embodiments. Non-limiting examples of non-polymeric materials can include certain metals, waxes, and organic materials (eg, organosilanes, perfluorinated or fluorinated organic materials).

Particles with reduced mucoadhesion

As described herein, in some embodiments, a method comprises identifying a material, such as a particle, for which it is desirable to reduce its mucoadhesive properties. Substances that require increased diffusivity through mucus, for example, may be hydrophobic, may have many hydrogen bond donors or acceptors, and/or may be highly charged. In some cases, the material may include a crystalline or amorphous solid material. A material capable of serving as a core may be coated with a suitable polymer described herein, thereby forming particles with a plurality of surface-altering agents on the surface, resulting in reduced mucoadhesion. Particles described herein as having reduced mucoadhesion are alternatively characterized as having increased transport through mucus, mobile in mucus, or mucus-penetrating (i.e., mucus-penetrating particles), which means that the particles have (Negative) means that the control particles are transported through the mucus faster than the control particles. The (negative) control particle can be a particle known to be mucoadhesive, eg, an unmodified particle or a core that is not coated with a coating described herein, such as a 200 nm carboxylated polystyrene particle.

In certain embodiments, the methods herein comprise preparing a pharmaceutical composition or formulation of a modified substance, e.g., in a formulation adapted for delivery (e.g., topical delivery) to a mucus or mucosal surface of a subject. do. A pharmaceutical composition having a surface modifying moiety may be delivered to a mucosal surface of a subject, cross a mucosal barrier in a subject, and/or may have prolonged retention, eg, due to reduced mucoadhesion, and/or , the uniform distribution of particles on the mucosal surface can be increased. As will be appreciated by those skilled in the art, mucus is a viscoelastic and adherent substance that traps most exogenous particles. Entrapped particles cannot reach the underlying epithelium and/or are rapidly cleared by the mucus clearance mechanism. In order for the particles to reach the underlying epithelium and/or for the particles to prolong their residence in the mucosal tissue, the particles must rapidly penetrate the mucus secretion and/or avoid the mucus clearance mechanism. If the particles do not substantially adhere to the mucus, the particles may be able to diffuse into the interstitial fluid between the mucin fibers and reach the underlying epithelium and/or not be removed by the mucin clearance mechanism. Thus, modifying a mucoadhesive material (e.g., a hydrophobic agent) with a material that reduces the mucoadhesion of particles may allow for efficient delivery of particles to the underlying epithelium and/or long-term retention at the mucosal surface. have.

Moreover, in some embodiments, the particles described herein with reduced mucoadhesion promote better distribution of the particles at the tissue surface and/or have a prolonged presence on the tissue surface as compared to the more tacky particles. For example, in some cases a luminal space, such as the gastrointestinal tract, is surrounded by a mucus-coated surface. Mucoadhesive particles delivered to this space are typically removed from the luminal space and from the mucus-coated surface by the body's natural clearance mechanism. Particles described herein with reduced mucoadhesion can remain in the luminal space for a relatively longer period of time compared to mucoadhesive particles. Such prolonged presence may prevent or reduce the clearance of particles and/or enable better distribution of particles on the tissue surface. Long-term presence can also affect particle transport through the luminal space, for example, particles can be distributed into the mucous layer and reach the underlying epithelium.

In certain embodiments, a material (eg, a core) coated with a polymer described herein crosses a mucus or mucosal barrier, exhibits prolonged retention, and/or increases the uniform distribution of particles on a mucosal surface in a subject. For example, such substances are cleared more slowly (eg, at least 2-fold, 5-fold, 10-fold, or even at least 20-fold more slowly) from the subject's body as compared to a (negative) control particle. The (negative) control particle can be a particle known to be mucoadhesive, eg, an unmodified particle or a core that is not coated with a coating described herein, such as a 200 nm carboxylated polystyrene particle.

In certain embodiments, the particles described herein have certain relative velocities, <V _mean > _relative , defined as:

<Equation 1>

where <V _mean > is the ensemble average trajectory-mean velocity, V _mean is the individual particle averaged over its trajectory, the sample is the particle of interest, and the negative control is the 200 nm carboxy Sylated polystyrene particles and positive controls are 200 nm polystyrene particles densely PEGylated with 2 kDa - 5 kDa PEG.

Relative velocity can be measured by multi-particle tracking techniques. For example, using a fluorescence microscope equipped with a CCD camera, 66.7 ms (15 frames/s) at 100x magnification from several regions within each sample for each type of particle: sample, negative control, and positive control. A 15 s movie can be captured at a temporal resolution of . Samples, negative and positive controls may be fluorescent particles observing traces. Alternatively, the non-fluorescent particles may be coated with a fluorescent molecule, a fluorescently tagged surface agent, or a fluorescently tagged polymer. Advanced image processing software (eg, Image Pro or MetaMorph) can be used to measure the individual trajectories of multiple particles over a time scale of 3.335 s (50 frames) or longer.

In some embodiments, the particles described herein are at least about 0.3, at least about 0.4, at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, at least about 1.0, at least about 1.1, at least about 1.2 in mucus. or greater, about 1.3 or greater, about 1.4 or greater, about 1.5 or greater, about 1.6 or greater, about 1.7 or greater, about 1.8 or greater, about 1.9 or greater, or about 2.0 or greater. In some embodiments, the particles described herein are about 10.0 or less, about 8.0 or less, about 6.0 or less, about 4.0 or less, about 3.0 or less, about 2.0 or less, about 1.9 or less, about 1.8 or less, about 1.7 or less, about 1.6 or less in mucus. or less, about 1.5 or less, about 1.4 or less, about 1.3 or less, about 1.2 or less, about 1.1 or less, about 1.0 or less, about 0.9 or less, about 0.8 or less, or about 1.7 or less. Combinations of the above-mentioned ranges are possible (eg, relative velocities of greater than or equal to about 0.5 and less than or equal to about 6.0). Other ranges are also possible. The mucus may be, for example, the mucus of the human cervix.

In certain embodiments, the particles described herein are mucus or mucosal barriers at a greater rate or diffusivity than control particles or corresponding particles (eg, unmodified and/or not coated with a coating described herein). can be spread through In some cases, the particles described herein are at least about 10 times, 20 times, 30 times, 50 times, 100 times, 200 times, 500 times, 1000 times, 2000 times, 5000 times, 10000 times greater than the control particles or corresponding particles. It can cross mucus or mucosal barriers at a rate of diffusion that is fold, or more, higher. In some cases, the particles described herein are no more than about 10000 times higher, no more than about 5000 times higher, no more than about 2000 times higher, no more than about 1000 times higher, no more than about 500 times higher than a control particle or corresponding particle. , mucus or mucosal barrier at a rate of diffusion as high as about 200 times or less, up to about 100 times as high, up to about 50 times as high, up to about 30 times as high, up to about 20 times as high, or up to about 10 times as high. can pass through Combinations of the above-mentioned ranges are also possible (eg, at least about 10 times and up to about 1000 times higher than a control particle or corresponding particle). Other ranges are also possible.

For purposes of comparison described herein, the corresponding particles may be approximately the same size, shape, and/or density as the test particles, but lack a coating that renders them mobile in mucus. In some cases, the measurement is based on a time scale of about 1 second, or about 0.5 seconds, or about 2 seconds, or about 5 seconds, or about 10 seconds. One of ordinary skill in the art would know how to measure geometric mean square displacement and diffusion rates.

Moreover, the particles described herein can be at least about 10 times, 20 times, 30 times, 50 times, 100 times, 200 times, 500 times, 1000 times, 2000 times, 5000 times, 10000 times, or It can cross mucus or mucosal barriers with an exceedingly high geometric mean square displacement. In some cases, the particles described herein are no more than about 10000 times higher, no more than about 5000 times higher, no more than about 2000 times higher, no more than about 1000 times higher, no more than about 500 times higher than a control particle or corresponding particle. , no more than about 200 times as high, no more than about 100 times as high, no more than about 50 times as high, no more than about 30 times as high, no more than about 20 times as high, or no more than about 10 times as high as a mucus with a geometric mean square displacement, or It can cross the mucosal barrier. Combinations of the above-mentioned ranges are also possible (eg, at least about 10 times and up to about 1000 times higher than a control particle or corresponding particle). Other ranges are also possible.

In some embodiments, the particles described herein diffuse through the mucosal barrier at a rate close to or at which the particles can diffuse through water. In some cases, the particles described herein have about 1/2 or less, about 1/4 or less, about 1/8 or less, about 1/16 or less, about 1/32 or less of the diffusivity that the particle diffuses through water under the same conditions. , about 1/50 or less, about 1/100 or less, about 1/200 or less, about 1/300 or less, about 1/400 or less, about 1/500 or less, about 1/600 or less, about 1/700 or less, about It may cross the mucosal barrier at a rate or diffusivity that is less than 1/800, less than about 1/900, less than about 1/1000, less than about 1/2000, less than about 1/5000, less than about 1/10,000. In some cases, the particles described herein have at least about 1/10,000, at least about 1/5000, at least about 1/2000, at least about 1/1000, at least about 1/900 of the diffusivity that the particle diffuses through water under the same conditions. , about 1/800 or more, about 1/700 or more, about 1/600 or more, about 1/500 or more, about 1/400 or more, about 1/300 or more, about 1/200 or more, about 1/100 or more, about It may cross the mucosal barrier at a rate or diffusivity of at least 1/50, at least about 1/32, at least about 1/16, at least about 1/8, at least about 1/4, or at least about 1/2. Combinations of the above-mentioned ranges are also possible (eg, greater than or equal to about 1/5000 and less than 1/500 of the diffusivity that a particle diffuses through water under the same conditions). Other ranges are also possible. The measurement may be based on a time scale of about 1 second, or about 0.5 seconds, or about 2 seconds, or about 5 seconds, or about 10 seconds.

In certain embodiments, the particles described herein are capable of diffusing through the mucus of the human cervix with a diffusivity that is about 1/500 or less of the diffusivity at which the particles diffuse through water. In some cases, the measurement is based on a time scale of about 1 second, or about 0.5 seconds, or about 2 seconds, or about 5 seconds, or about 10 seconds.

In certain embodiments, the present invention provides particles that migrate through mucus, such as the mucus of the human cervix, with a certain absolute diffusivity. For example, the particles described herein can be at least about 1 x 10 ^-4 μm/s, 2 x 10 ^-4 μm/s, 5 x 10 ^-4 μm/s, 1 x 10 ^-3 μm/s, 2 x 10 ^-3 μm/s, 5 x 10 ^-3 μm/s, 1 x 10 ^-2 μm/s, 2 x 10 ^-2 μm/s, 4 x 10 ^-2 μm/s, 5 x 10 ^-2 μm/s , 6 x 10 ^-2 μm/s, 8 x 10 ^-2 μm/s, 1 x 10 ^-1 μm/s, 2 x 10 ^-1 μm/s, 5 x 10 ^-1 μm/s, 1 μm/s , or with a diffusivity of 2 μm/s. In some cases, the particles are about 2 μm/s or less, about 1 μm/s or less, about 5×10 ⁻¹ μm/s or less, about 2×10 ⁻¹ μm/s or less, about 1×10 ⁻¹ μm/s or less s or less, about 8 x 10 ^-2 μm/s or less, about 6 x 10 ^-2 μm/s or less, about 5 x 10 ^-2 μm/s or less, about 4 x 10 ^-2 μm/s or less, about 2 x 10 ^-2 μm/s or less, about 1 x 10 ^-2 μm/s or less, about 5 x 10 ^-3 μm/s or less, about 2 x 10 ^-3 μm/s or less, about 1 x 10 ^-3 μm/s or less or less, about 5×10 ⁻⁴ μm/s or less, about 2×10 ⁻⁴ μm/s or less, or about 1×10 ⁻⁴ μm/s or less. Combinations of the aforementioned ranges are also possible (eg, ^{greater than or equal to about 2 x 10 -4} μm/s and less than or equal to ^{about 1 x 10 -1 μm/s).} Other ranges are also possible. In some cases, the measurement is based on a time scale of about 1 second, or about 0.5 seconds, or about 2 seconds, or about 5 seconds, or about 10 seconds.

It should be noted that although many of the mobility (eg, relative velocity, diffusivity) described herein can be measured in human cervical mucus, they can likewise be measured in other types of mucus.

In certain embodiments, the particles described herein comprise surface modifying moieties at a predetermined density. The surface-altering moiety can be a portion of the surface-altering agent, for example, exposed to a solvent containing the particle. As an example, the PEG segment may be a surface modifying moiety of the surface modifying agent PEG-PPO-PEG. In some cases, the surface modifying moiety and/or surface modifying agent is at least about 0.001 units or molecules per nm, at least about 0.002 per nm, at least about 0.005, at least about 0.01, at least about 0.02, at least about 0.05, at least about 0.1 or more, about 0.2 or more, about 0.5 or more, about 1 or more, about 2 or more, about 5 or more, about 10 or more, about 20 or more, about 50 or more, about 100 or more units or molecules, or more units per nm2, or It exists in molecular density. In some cases, the surface modifying moiety and/or surface modifying agent has about 100 units or molecules per nm2 or less, about 50 or less, about 20 or less, about 10 or less, about 5 or less, about 2 or less, about It is present in a density of units or molecules of 1 or less, about 0.5 or less, about 0.2 or less, about 0.1 or less, about 0.05 or less, about 0.02 or less, or about 0.01 or less. Combinations of the above-mentioned ranges are also possible (eg, a density of about 0.01 or more and about 1 or less units or molecules per nm 2 ). Other ranges are also possible. In some embodiments, the density values described above may be average densities because the surface-altering agent is in equilibrium with the other components in solution.

One of ordinary skill in the art would know how to quantify or estimate the average density of surface altering moieties (see, e.g., SJ Budijono et al., Colloids and Surfaces A: Physicochem. Eng. Aspects 360 (2010) 105-110) and See Joshi, et al ., Anal. Chim. Acta 104 (1979) 153-160, each of which is incorporated herein by reference). For example, as described herein, the average density of surface modifying moieties can be determined using HPLC quantification and DLS analysis. A suspension of particles of interest for surface density determination is first sized using DLS: a small volume is diluted to an appropriate concentration (e.g., ˜100 μg/mL) and the z-average diameter is taken as a representative measure of particle size. do. The remaining suspension is then divided into two aliquots. Using HPLC, the first aliquot is assayed for total concentration of core material and total concentration of surface modifying moieties. A second aliquot is assayed for the concentration of free or unbound surface modifying moieties again using HPLC. In order to obtain only free or unbound surface modifying moieties from the second aliquot, the particles, and thus any bound surface modifying moieties, are removed by ultracentrifugation. By subtracting the concentration of unbound surface modifying moieties from the total concentration of surface modifying moieties, the concentration of bound surface modifying moieties can be determined. Also, since the total concentration of the core material was determined from the first aliquot, the mass ratio of the core material to the surface modifying moiety can be determined. The molecular weight of the surface modifying moieties can be used to calculate the number of surface modifying moieties relative to the mass of the core material. To convert this number to a surface density measurement, it is necessary to calculate the surface area per mass of the core material. The volume of the particle is approximated as the volume of a sphere having a diameter obtained from DLS, making it possible to calculate the surface area per mass of the core material. In this way the number of surface modifying moieties per surface area can be determined. Unless otherwise specified, density values herein were determined using this method.

Density also quantifies or estimates the core material surface area by methods such as electron microscopy, light scattering, or surface interaction measurements, and then quantifies the attached surface agent(s) by methods such as liquid chromatography or mass spectrometry. can be measured by (See, eg, Wang et al., Angew Chem Int Ed Engl , 2008, 47(50), 9726-9, which is incorporated herein by reference).

In certain embodiments, the particles described herein may include surface-altering moieties and/or surface-altering agents that affect the zeta potential of the particle. The zeta potential of the coated particles can be, for example, about -100 mV or more, about -75 mV or more, about -50 mV or more, about -40 mV or more, about -30 mV or more, about -20 mV or more, about -10 mV or greater, about -5 mV or greater, about 5 mV or greater, about 10 mV or greater, about 20 mV or greater, about 30 mV or greater, about 40 mV or greater, about 50 mV or greater, about 75 mV or greater, or about 100 mV or greater have. Combinations of the above-mentioned ranges are possible (eg, a zeta potential of greater than or equal to about -50 mV and less than or equal to about 50 mV). Other ranges are also possible.

The coated particles described herein may have any suitable shape and/or size. In some embodiments, the coated particles have a shape substantially similar to that of the core. In some cases, the coated particles described herein can be nanoparticles, i.e., the particles have a characteristic dimension of about 1 micrometer or less, wherein the characteristic dimension of the particle is the diameter of a perfect sphere having the same volume as the particle. In other embodiments, larger sizes are possible (eg, about 1-10 microns). The plurality of particles may also be characterized, in some embodiments, by an average size (eg, an average maximum cross-sectional dimension, or an average minimum cross-sectional dimension for the plurality of particles). The plurality of particles are, for example, about 10 μm or less, about 5 μm or less, about 1 μm or less, about 800 nm or less, about 700 nm or less, about 500 nm or less, 400 nm or less, 300 nm or less, about 200 nm or less , about 100 nm or less, about 75 nm or less, about 50 nm or less, about 40 nm or less, about 35 nm or less, about 30 nm or less, about 25 nm or less, about 20 nm or less, about 15 nm or less, or about 5 nm or less It may have the following average size. In some cases, the plurality of particles are, for example, about 5 nm or more, about 20 nm or more, about 50 nm or more, about 100 nm or more, about 200 nm or more, about 300 nm or more, about 400 nm or more, about 500 nm or more. It may have an average size of at least about 1 μm, or about 5 μm or more. Combinations of the above-mentioned ranges are also possible (eg, an average size of greater than or equal to about 50 nm and less than or equal to about 500 nm). Other ranges are also possible. In some embodiments, the sizes of the cores formed by the methods described herein have a Gaussian distribution.

drugs

In some embodiments, the coated particles comprise one or more agents. The agent may be present in the core of the particle and/or present in the coating of the particle (eg, dispersed throughout the core and/or coating). In some cases, the medicament may be disposed on the surface of the particle (eg, on the outer surface of the coating, on the inner surface of the coating, on the surface of the core). An agent can often be contained within and/or placed within a portion of a particle using known techniques (eg, coating, adsorption, covalent linking, or other processes). In some cases, the agent may be present in the core of the particle prior to or during coating of the particle. In some cases, the agent is present during formation of the core of the particle, as described herein.

Non-limiting examples of pharmaceuticals include imaging agents, diagnostic agents, therapeutic agents, agents with detectable labels, nucleic acids, nucleic acid analogs, small molecules, peptidomimetics, proteins, peptides, lipids, vaccines, viral vectors, viruses, and surfactants. do.

In some embodiments, the medicament contained in the particles described herein has a therapeutic, diagnostic, or imaging effect in a targeted mucosal tissue. Non-limiting examples of mucosal tissues include oral cavity (including, for example, buccal and esophageal membranes and tonsil surfaces), eye, gastrointestinal tract (including, for example, stomach, small intestine, large intestine, colon, rectum), nasal cavity, respiratory tract (eg, , including nasal, pharyngeal, tracheal and bronchial membranes), and genital (eg, vaginal, cervical, and urethral membranes) tissues.

Any suitable number of agents may be present in the particles described herein. For example, at least 1, at least 2, at least 3, at least 4, at least 5, or more, but generally less than 10 agents may be present in a particle described herein.

A number of drugs that are mucoadhesive are known in the art and can be used as pharmaceuticals in the particles described herein (see, e.g., Khanvilkar K, Donovan MD, Flanagan DR, Drug transfer through mucus, Advanced Drug Delivery Reviews 48 (2001) 173-193];[Bhat PG, Flanagan DR, Donovan MD. Drug diffusion through cystic fibrotic mucus: steady-state permeation, rheologic properties, and glycoprotein morphology, J Pharm Sci, 1996 Jun;85(6):624 -30]). Further non-limiting examples of medicaments include imaging and diagnostic agents (such as radiopaque agents, labeled antibodies, labeled nucleic acid probes, dyes such as colored or fluorescent dyes, etc.) and adjuvants (radiosensitizers, transfection enhancers, chemoattractants and chemoattractants, peptides that modulate cell adhesion and/or cell motility, cell penetrants, vaccine potentiators, multidrug resistance and/or inhibitors of efflux pumps, etc.) do.

Further non-limiting examples of medicaments include alloxiprine, auranofin, azapropazone, berorylate, diflunisal, etodolac, fenbufen, fenoprofen calsim, flurbiprofen, furosemide, ibuprofen , indomethacin, ketoprofen, loteprednol etabonate, meclofenamic acid, mefenamic acid, nabumetone, naproxen, oxyphenbutazone, phenylbutazone, piroxan, sulindac, albendazole, be Phenium hydroxynaphthoate, cambendazole, dichlorophen, ivermectin, mebendazole, oxamniquine, oxpendazole, oxantel embonate, praziquantel, pyrantel embonate, thiabendazole, amiodarone HCl , disopyramide, flecainide acetate, quinidine sulfate. Antibacterial agents: Benetamine penicillin, synoxacin, ciprofloxacin HCl, clarithromycin, clofazimine, cloxacillin, demeclocycline, doxycycline, erythromycin, ethionamide, imipenem, nalidixic acid, nitrofuran Toin, rifampicin, spiramicin, sulfabenzamide, sulfadoxine, sulfamerazine, sulfacetamide, sulfadiazine, sulfafurazole, sulfamethoxazole, sulfapyridine, tetracycline, trimethoprim, dicoumarol, dipypi lidamole, nicumalone, phenindione, amoxapine, maprotiline HCl, myanserine HCL, nortriptyline HCl, trazodone HCL, trimipramine maleate, acetohexaamide, chlorpropamide, glibenclamide , gliclazide, glipizide, tolazamide, tolbutamide, beclamide, carbamazepine, clonazepam, etotoin, methoin, metsuccibead, methylphenobarbitone, oxcarbazepine , paramethadione, phenasemide, phenobarbitone, phenytoin, phensuccimide, primidone, sulthiam, valproic acid, amphotericin, butoconazole nitrate, clotrimazole, econazole nitrate, fluconazole, flu Cytosine, griseofulvin, itraconazole, ketoconazole, miconazole, natamycin, nystatin, sulconazole nitrate, terbinafine HCl, terconazole, thioconazole, undecenoic acid, allopurinol, propeneside, sulfin -pyrazone, amlodipine, benidipine, darodipine, dilitazem HCl, diazoxide, felodipine, guanabenz acetate, isradipine, minoxidil, nicardipine HCl, nifedipine, nimodipine, phenoxybenzamine HCl , prazosin HCL, reserpine, terazosin HCL, amodiaquine, chloroquine, chlorproguanil HCl, halopanthrine HCl, mefloquine HCl, loguanil HCl, pyrimethamine, quinine sulfate, dihydroergotamine mesyl Late, Ergotamine Tartrate, Methisergide Maleate, Pyzotifen Maleate, Sumatriptan Succinate, Atropine, Benzhexol HCl, Biperidene, Etopropazine HCl, Hyosyamine, Mepenzolate Bromide, Oxyphencycline HCl, tropicamide, aminoglutethimide, amsacrine, azathioprine, bu sulfan, chlorambucil, cyclosporine, dacarbazine, estramustine, etoposide, lomustine, melphalan, mercaptopurine, methotrexate, mitomycin, mitophan, mitoxantrone, procarbazine HCl, tamoxifen citrate , testolactone, benznidazole, clioquinol, decoquinate, diiodohydroxyquinoline, dioxanide furoate, dinitolmide, purazolidone, metronidazole, nimorazole, nitrofurazone, ornidazole, tinidazole sol, carbimazole, propylthiouracil, alprazolam, amylobarbitone, barbitone, bentazepam, bromazepam, bromperidol, brotisolam, butobarbitone, carbromal, chlordiazepoxide, Chlormethiazole, Chlorpromazine, Clovazam, Clotiazepam, Clozapine, Diazepam, Dropperidol, Etinamate, Pullunanisone, Flunitrazepam, Fluopramazine, Flufenthixol Decanoate, Fluphenazine De canoate, flurazepam, haloperidol, lorazepam, lormetazepam, medazepam, meprovamate, methaqualone, midazolam, nitrazepam, oxazepam, pentobarbitone, perphenazine pimozide, prochlorperazine, Sulpyride, temazepam, thioridazine, triazolam, zopiclone, acebutolol, alprenolol, atenolol, labetalol, metoprolol, nadolol, oxprenolol, pindolol, propanolol, amrinone, digitoxin , digoxin, enoxymon, lanatoside C, medigoxin, beclomethasone, betamethasone, budesonide, cortisone acetate, desoxymethasone, dexamethasone, fludrocortisone acetate, flunisolide, flucortolone, fluticasone pro Cypionate, hydrocortisone, methylprednisolone, prednisolone, prednisone, traamcinolone, acetazolamide, amiloride, bendrofluazide, bumetanide, chlorothiazide, chlorthalidone, etacric acid, pruce Mead, metolazone, spironolactone, triamterene, bromocriptine mesylate, risuride maleate, bisacodyl, cimetidine, cisapride, diphenoxylate HCl, domperidone, famotidine, loperamide, mesala Gin, nizatidine, omeprazole, ondansetron HCL, ranitidine HCl, sulfasalazine, acri Bastin, Astemizole, Cinnarizine, Cyclizine, Cyproheptadiene HCl, Dimenhydrinate, Pullunarizine HCl, Loratadine, Meclozine HCl, Oxatomide, Terfenadine, Bennafibrate, Clofibrate , fenofibrate, gemfibrozil, probucol, amyl nitrate, glyceryl trinitrate, isosorbide dinitrate, isosorbide mononitrate, pentaerythritol tetranitrate, beta-carotene, vitamin A, vitamin B2, Vitamin D, vitamin E, vitamin K, codeine, dextropropioxifene, diamorphine, dihydrocodeine, meptazinol, methadone, morphine, nalbuphine, pentazocine, clomiphene citrate, danazole, ethinyl estradiol, medroxy progesterone acetate, mestranol, methyltestosterone, norethisterone, norgestrel, estradiol, conjugated estrogen, progesterone, stanozolol, stivestrol, testosterone, tibolone, amphetamine, dexamphetamine, dexfenfluramine, fenfluramine, and margin stone.

Uses and pharmaceutical compositions

The particles described herein can be used in any suitable application. In some cases, the particle is a part of the pharmaceutical composition (eg, as described herein), for example, through or onto a mucus or mucosal surface, an agent (eg, drug, therapeutic agent, diagnostic agent, imaging agent). It is used to convey A pharmaceutical composition may comprise one or more particles described herein and one or more pharmaceutically acceptable excipients or carriers. The composition may be used to treat, prevent, and/or diagnose a condition in a subject, wherein the method comprises administering to the subject the pharmaceutical composition. A subject or patient treated by the articles and methods described herein can mean a human or non-human animal, such as primates, mammals, and vertebrates.

Methods related to treating a subject include preventing the disease, disorder, or condition from developing in a subject who may be susceptible to the disease, disorder and/or condition but has not yet been diagnosed as having the disease; inhibiting the disease, disorder or condition, eg, delaying its progression; and alleviating the disease, disorder, or condition, eg, causing regression of the disease, disorder, and/or condition. Treating a disease or condition includes ameliorating one or more symptoms of a particular disease or condition, even if the underlying pathophysiology is not affected (e.g., although the analgesic does not treat the cause of the pain, such analgesic) such treatment of a subject's pain by administering

In some embodiments, a pharmaceutical composition described herein can be delivered to and cross a mucosal barrier (e.g., mucus) in a subject and/or a mucosal surface, e.g., due to reduced mucoadhesion. may exhibit prolonged retention and/or increased uniform distribution of particles in the Non-limiting examples of mucosal tissues include oral cavity (including, for example, buccal and esophageal membranes and tonsil surfaces), eye, gastrointestinal tract (including, for example, stomach, small intestine, large intestine, colon, rectum), nasal cavity, respiratory tract (eg, , including nasal, pharyngeal, tracheal and bronchial membranes), genital (eg, vaginal, cervical and urethral membranes).

Pharmaceutical compositions described herein and for use in accordance with the articles and methods described herein may include pharmaceutically acceptable excipients or carriers. A pharmaceutically acceptable excipient or pharmaceutically acceptable carrier may include any suitable type of non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulating aid. Some examples of substances that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oil, peanut oil, cottonseed oil; safflower oil; Sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffers, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, mold release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants, as well as in the judgment of the formulator. may be present in the composition. As will be appreciated by one of ordinary skill in the art, excipients may be selected based on the route of administration, the agent delivered, the time course of delivery of the agent, and the like, as described below.

Pharmaceutical compositions containing the particles described herein can be administered to a subject via any route known in the art. These are oral, sublingual, nasal, intradermal, subcutaneous, intramuscular, rectal, vaginal, intravenous, intraarterial, intracisternal, intraperitoneal, intravitreal, periocular, topical (as a powder, cream, ointment, or drop); including, but not limited to, buccal and inhalation administration. In some embodiments, the compositions described herein can be administered parenterally as injection (intravenous, intramuscular, or subcutaneous), drop infusion formulations, or suppositories. As will be appreciated by one of ordinary skill in the art, the route of administration and effective dosage to achieve the desired biological effect will be determined by the agent administered, the target organ, the agent administered, the time course of administration, the disease being treated, the intended use, and the like. can

For example, the particles may be included in a pharmaceutical composition formulated as a nasal spray such that the pharmaceutical composition is delivered across the mucus layer of the nose. As another example, the particles may be incorporated into a pharmaceutical composition that is formulated as an inhaler such that the pharmaceutical composition is delivered across the mucous layer of the lung. As another example, when the composition is administered orally, it may be formulated as a tablet, capsule, granule, powder, or syrup. Similarly, the particles may be incorporated into pharmaceutical compositions for delivery via the ocular, gastrointestinal, nasal, respiratory, rectal, urethral, and/or vaginal tissues.

For application by the ophthalmic mucosal route, the subject compositions may be formulated as eye drops or ophthalmic ointments. These formulations may be prepared in a conventional manner and, if desired, the subject compositions may be mixed with any conventional additives such as buffers or pH-adjusting agents, tonicity adjusting agents, viscosity adjusting agents, suspension stabilizers, preservatives, and other pharmaceutical excipients. can Moreover, in certain embodiments, the subject compositions described herein may be lyophilized or subjected to another suitable drying technique, such as spray drying.

In some embodiments, the particles described herein that can be administered as an inhalant or an aerosol formulation include one or more agents useful in inhalation therapy, such as adjuvants, diagnostic agents, imaging agents, or therapeutic agents. The particle size of the particulate medicament should be such that substantially all of the medicament can be inhaled into the lung upon administration of the aerosol formulation, for example about 20 micrometers or less, for example from about 1 to about 10 micrometers, for example about It can range from 1 to about 5 micrometers, although other ranges are also possible. The particle size of the medicament can be reduced in a conventional manner, for example by milling or micronisation. Alternatively, the particulate medicament may be administered to the lungs by nebulization of the suspension. The final aerosol formulation contains, for example, 0.005-90% w/w, 0.005-50%, 0.005-10%, about 0.005-5% w/w, or 0.01-1.0% w/w of the drug relative to the total amount of the formulation. can do. Other ranges are also possible.

Although never required, it is preferred that the formulations described herein do not contain any components that can cause decomposition of stratospheric ozone. In particular, in some embodiments, a propellant is selected that does not contain or consist essentially of _{chlorofluorocarbons such as CCl 3} F, CCl ₂ F ₂ , and CF ₃ CCl _{3 .}

The aerosol may contain a propellant. The propellant may optionally contain an adjuvant having a higher polarity and/or a higher boiling point than the propellant. Polar adjuvants that may be used include (eg, C _2-6 ) aliphatic alcohols and polyols such as ethanol, isopropanol, and propylene glycol, preferably ethanol. In general, only small amounts of a polar adjuvant (eg, 0.05-3.0% w/w) may be needed to improve the stability of the dispersion - use of amounts greater than 5% w/w tends to dissolve the drug This can be. Formulations according to embodiments described herein may contain less than 1% w/w of a polar adjuvant, for example about 0.1% w/w. However, the formulations described herein may be substantially free of polar adjuvants, particularly ethanol. Suitable volatile adjuvants include saturated hydrocarbons such as propane, n-butane, isobutane, pentane and isopentane and alkyl ethers such as dimethyl ether. In general, up to 50% w/w of the propellant may include 1 to 30% w/w of a _{volatile adjuvant, for example volatile saturated C 1} -C _{6 hydrocarbons.} Optionally, the aerosol formulation according to the invention may further comprise one or more surfactants. The surfactant is physiologically acceptable when administered by inhalation. Surfactants within this category, such as L-α-phosphatidylcholine (PC), 1,2-dipalmitoylphosphatidylcholine (DPPC), oleic acid, sorbitan trioleate, sorbitan mono-oleate, sorbitan monolaurate, poly Oxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, natural lecithin, oleyl polyoxyethylene ether, stearyl polyoxyethylene ether, lauryl polyoxyethylene ether, block copolymer of oxyethylene and oxypropylene , synthetic lecithin, diethylene glycol dioleate, tetrahydrofurfuryl oleate, ethyl oleate, isopropyl myristate, glyceryl monooleate, glyceryl monostearate, glyceryl monoricinolate, cetyl alcohol, stearate aryl alcohol, polyethylene glycol 400, cetyl pyridinium chloride, benzalkonium chloride, olive oil, glyceryl monolaurate, corn oil, cottonseed oil, and sunflower seed oil.

The formulations described herein may be prepared by dispersing the particles in a selected propellant and/or co-propellant in an appropriate container, utilizing, for example, sonication. The particles can be suspended in the co-propellant and filled into a suitable container. The valve of the vessel is then closed in place and the propellant is introduced by pressure filling through the valve in a conventional manner. The particles can thus be suspended or dissolved in a liquefied propellant, sealed in a container equipped with a metering valve and fitted with an actuator. Such metered dose inhalers are well known in the art. The metering valve can meter 10 to 500 μL, preferably 25 to 150 μL. In certain embodiments, dispersion may be achieved using a dry powder inhaler (eg, a spinhaler) for the particles (remaining as a dry powder). In other embodiments, the nanospheres can be suspended in an aqueous fluid and atomized into microdroplets to aerosolize into the lungs.

Ultrasonic nebulizers can be used because they minimize exposure of the agent to shear, which can result in particle degradation. Usually, aqueous aerosols are prepared by formulating an aqueous solution or suspension of the particles with conventional pharmaceutically acceptable carriers and stabilizers/surface-altering agents. Carriers and stabilizers/surface modifiers depend on the requirements of the particular composition, but typically include nonionic surfactants (Tween, Pluronic ^® , or polyethylene glycol), harmless proteins such as serum albumin, sorbitan esters, oleic acid. , lecithin, amino acids such as glycine, buffers, salts, sugars, or sugar alcohols. Aerosols are generally prepared from isotonic solutions.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient (i.e., microparticles, nanoparticles, liposomes, micelles, polynucleotide/lipid complexes), liquid dosage forms may contain inert diluents commonly used in the art, for example, water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, fatty acid esters of polyethylene glycol and sorbitan, and mixtures thereof, and the like. In addition to inert diluents, oral compositions may also include adjuvants such as emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be as a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. Any bland fixed oil may be used for this purpose, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. In certain embodiments, the particles are suspended in a carrier fluid comprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) Tween 80.

Formulations for injection may be sterilized, for example, by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use, or by filtration through a bacteria-retaining filter. can

Compositions for rectal or vaginal administration may be suppositories which may be prepared by mixing the particles with a suitable non-irritating excipient or carrier such as cocoa butter, polyethylene glycol, or a suppository wax, which is solid at ambient temperature but liquid at body temperature and thus rectal. or melt in the vaginal cavity, releasing particles.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the particles may contain one or more inert, pharmaceutically acceptable excipients or carriers such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and silicic acid, b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, etc., c) wetting agents such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glycerol monostearate and the like, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. . In the case of capsules, tablets and pills, the dosage form may also include a buffer.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition which releases the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric materials and waxes.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

Dosage forms for topical or transdermal administration of the pharmaceutical compositions of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The particles are admixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservatives or buffers as may be required. Ophthalmic formulations, ear drops, and eye drops are also contemplated as being within the scope of this invention.

Ointments, pastes, creams, and gels may contain, in addition to the particles described herein, excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or a mixture thereof.

Powders and sprays may contain, in addition to the particles described herein, excipients such as lactose, talc, silicic acid, aluminum oxide, calcium silicate, and polyamide powder, or mixtures of these substances. Sprays may also contain customary propellants, such as chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlled delivery of compounds to the body. Such dosage forms can be prepared by dissolving or dispersing the microparticles or nanoparticles in a suitable medium. Absorption enhancers may also be used to increase the flux of the compound across the skin. The rate can be controlled by providing a rate controlling membrane or by dispersing the particles in a polymer matrix or gel.

The particles described herein, including medicaments, can be administered to a subject to be delivered in an amount sufficient to deliver a therapeutically effective amount of the incorporated medicament to the subject as part of a diagnostic, prophylactic, or therapeutic treatment. In general, an effective amount of a drug or ingredient refers to the amount necessary to elicit a desired biological response. The desired concentration of the drug in the particle depends on a number of factors including, but not limited to, the rate of absorption, inactivation, and excretion of the drug, as well as the rate of delivery of the compound from the subject composition, the desired biological endpoint, the agent to be delivered, the target tissue, and the like. will be determined by It should be noted that dosage values may also vary depending on the severity of the condition to be alleviated. It should be further understood that, for any particular subject, the particular dosage regimen should be adjusted over time according to the individual needs and the professional judgment of the person supervising or administering the administration of the composition. Typically, dosing will be determined using techniques known to those skilled in the art.

The agent may be present in any suitable amount, for example, at least about 0.01 wt%, at least about 0.1 wt%, at least about 1 wt%, at least about 5 wt%, at least about 10 wt%, at least about 20 wt% of the composition and/or formulation. More than one may be present in the composition and/or formulation. In some cases, the agent is present in the composition and/or formulation in about 30 wt% or less, about 20 wt% or less, about 10 wt% or less, about 5 wt% or less, about 2 wt% or less, or about 1 wt% or less. can Combinations of the above-mentioned ranges are also possible (eg, present in an amount greater than or equal to about 0.1 wt % and less than or equal to about 10 wt %). Other ranges are also possible. In certain embodiments, the agent is about 0.1-2 wt % of the composition and/or formulation. In certain embodiments, the agent is about 2-20 wt % of the composition and/or formulation. In certain embodiments, the agent is about 0.2 wt % of the composition and/or formulation. In certain embodiments, the agent is about 0.4 wt % of the composition and/or formulation. In certain embodiments, the agent is about 1 wt % of the composition and/or formulation. In certain embodiments, the agent is about 2 wt % of the composition and/or formulation. In certain embodiments, the agent is about 5 wt % of the composition and/or formulation. In certain embodiments, the agent is about 10 wt % of the composition and/or formulation.

The concentration and/or amount of any agent administered to a subject can be readily determined by one of ordinary skill in the art. Known methods are also available for assaying local tissue concentration, rate of diffusion from particles and local blood flow before and after administration of a therapeutic agent.

The compositions and/or formulations described herein may have any suitable osmolarity. In some embodiments, the compositions and/or formulations described herein are at least about 0 mOsm/L, at least about 5 mOsm/L, at least about 25 mOsm/L, at least about 50 mOsm/L, at least about 75 mOsm/L, about It may have an osmolality of 100 mOsm/L or more, about 150 mOsm/L or more, about 200 mOsm/L or more, about 250 mOsm/L or more, or about 310 mOsm/L or more. In certain embodiments, the compositions and/or formulations described herein are about 310 mOsm/L or less, about 250 mOsm/L or less, about 200 mOsm/L or less, about 150 mOsm/L or less, about 100 mOsm/L or less, about It may have an osmolality of 75 mOsm/L or less, about 50 mOsm/L or less, about 25 mOsm/L or less, or about 5 mOsm/L or less. Combinations of the above-mentioned ranges are also possible (eg, osmolarity of greater than or equal to about 0 mOsm/L and less than or equal to about 50 mOsm/L). Other ranges are also possible. The osmolarity of the composition and/or formulation can be varied, for example, by varying the concentration of salt present in the solvent of the composition and/or formulation.

In one set of embodiments, the composition and/or formulation comprises one or more chelating agents. Chelating agent, as used herein, refers to a chemical compound that has the ability to react with a metal ion to form a complex through one or more bonds. The one or more bonds are typically ionic or coordination bonds. The chelating agent may be an inorganic or organic compound. Metal ions capable of catalyzing certain chemical reactions (eg, oxidation reactions) may lose their catalytic activity when the metal ion binds to a chelating agent to form a complex. Thus, a chelating agent may exhibit preservation properties when it binds to a metal ion. Any suitable chelating agent having preservative properties can be used, such as phosphonic acids, aminocarboxylic acids, hydroxycarboxylic acids, polyamines, aminoalcohols, and polymeric chelating agents. Specific examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentacetic acid (DTPA), N -hydroxyethylethylene diaminetriacetic acid (HEDTA), tetraborate, triethylamine diamines, and salts and derivatives thereof. In certain embodiments, the chelating agent is EDTA. In certain embodiments, the chelating agent is a salt of EDTA. In certain embodiments, the chelating agent is disodium EDTA.

The chelating agent may be present in a concentration suitable for compositions and/or formulations comprising the coated particles described herein. In certain embodiments, the concentration of the chelating agent is at least about 0.0003 wt%, at least about 0.001 wt%, at least about 0.003 wt%, at least about 0.01 wt%, at least about 0.03 wt%, at least about 0.05 wt%, at least about 0.1 wt% or more, about 0.3 wt % or more, about 1 wt % or more, or about 3 wt % or more. In certain embodiments, the concentration of the chelating agent is about 3 wt% or less, about 1 wt% or less, about 0.3 wt% or less, about 0.1 wt% or less, about 0.05 wt% or less, about 0.03 wt% or less, about 0.01 wt% or less or less, about 0.003 wt % or less, about 0.001 wt % or less, or about 0.0003 wt % or less. Combinations of the above recited ranges are possible (eg, a concentration of at least about 0.01 wt % and up to about 0.3 wt %). Other ranges are also possible. In certain embodiments, the concentration of the chelating agent is about 0.001-0.1 wt %. In certain embodiments, the concentration of the chelating agent is about 0.005 wt %. In certain embodiments, the concentration of the chelating agent is about 0.01 wt %. In certain embodiments, the concentration of the chelating agent is about 0.05 wt %. In certain embodiments, the concentration of the chelating agent is about 0.1 wt %.

In some embodiments, the chelating agent may be present in the composition and/or formulation in one or more of the aforementioned ranges during the forming and/or dilution processes described herein. In certain embodiments, the chelating agent may be present in the composition and/or formulation in one or more of the aforementioned ranges in the final product.

In some embodiments, antimicrobial agents may be included in compositions and/or formulations comprising the coated particles described herein. As used herein, antimicrobial agent refers to a bioactive agent that is effective in inhibiting, preventing, or protecting against microorganisms, such as bacteria, microorganisms, fungi, viruses, spores, yeast, molds, and the like, generally associated with infection. Examples of antimicrobial agents include cephalosporins, clindamycin, chloramphenicol, carbapenem, minocycline, lymphapine, penicillin, monobactam, quinolones, tetracyclines, macrolides, sulfa antibiotics, trimethoprim, fu Cidic acid, aminoglycoside, amphotericin B, azole, flucytosine, cilofulzine, bactericidal nitrofuran compound, metallic silver or nanoparticles of silver alloy containing about 2.5 wt % copper, silver citrate, silver acetate, benzoic acid silver, bismuth pyrithione, zinc pyrithione, zinc percarbonate, zinc perborate, bismuth salts, parabens (eg, methyl-, ethyl-, propyl-, butyl-, and octyl-benzoic acid esters), citric acid, benzal conium chloride (BAC), lymphamycin, and sodium percarbonate.

The antimicrobial agent may be present in a concentration suitable for compositions and/or formulations comprising the coated particles described herein. In certain embodiments, the concentration of the antimicrobial agent is at least about 0.0003 wt%, at least about 0.001 wt%, at least about 0.003 wt%, at least about 0.01 wt%, at least about 0.03 wt%, at least about 0.1 wt%, at least about 0.3 wt% or more, about 1 wt% or more, or about 3 wt% or more. In certain embodiments, the concentration of the antimicrobial agent is about 3 wt% or less, about 1 wt% or less, about 0.3 wt% or less, about 0.1 wt% or less, about 0.03 wt% or less, about 0.01 wt% or less, about 0.003 wt% or less or less, about 0.001 wt % or less, or about 0.0003 wt % or less. Combinations of the above-mentioned ranges are possible (eg, a concentration of at least about 0.001 wt % and up to about 0.1 wt %). Other ranges are also possible. In certain embodiments, the concentration of the antimicrobial agent is about 0.001-0.05 wt %. In certain embodiments, the concentration of the antimicrobial agent is about 0.002 wt %. In certain embodiments, the concentration of the antimicrobial agent is about 0.005 wt %. In certain embodiments, the concentration of the antimicrobial agent is about 0.01 wt %. In certain embodiments, the concentration of the antimicrobial agent is about 0.02 wt %. In certain embodiments, the concentration of the antimicrobial agent is about 0.05 wt %.

In some embodiments, the antimicrobial agent may be present in the composition and/or formulation in one or more of the aforementioned ranges during the forming and/or dilution processes described herein. In certain embodiments, the antimicrobial agent may be present in the composition and/or formulation in one or more of the aforementioned ranges in the final product.

In some embodiments, isotonic agents may be included in compositions and/or formulations comprising the coated particles described herein. Tonicity agent, as used herein, refers to a compound or substance that can be used to adjust the composition of an agent to a desired osmolarity range. In certain embodiments, the desired osmolarity range is an isotonic range compatible with blood. In certain embodiments, the desired osmolarity range is hypotonic. In certain embodiments, the desired osmolarity range is hypertonic. Examples of isotonic agents include glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol, salt-sodium citrate (SSC), and the like. In certain embodiments, a combination of one or more isotonic agents may be used. In certain embodiments, the isotonic agent is glycerin. In certain embodiments, the isotonic agent is sodium chloride.

Tonicity agents (such as those described herein) may be present in concentrations suitable for compositions and/or formulations comprising the coated particles described herein. In certain embodiments, the concentration of the tonicity agent is at least about 0.003 wt%, at least about 0.01 wt%, at least about 0.03 wt%, at least about 0.1 wt%, at least about 0.3 wt%, at least about 1 wt%, at least about 3 wt% or more, about 10 wt% or more, about 20 wt% or more, or about 30 wt% or more. In certain embodiments, the concentration of the isotonic agent is about 30 wt% or less, about 10 wt% or less, about 3 wt% or less, about 1 wt% or less, about 0.3 wt% or less, about 0.1 wt% or less, about 0.03 wt% or less or less, about 0.01 wt % or less, or about 0.003 wt % or less. Combinations of the above-mentioned ranges are possible (eg, a concentration of at least about 0.1 wt % and up to about 10 wt %). Other ranges are also possible. In certain embodiments, the concentration of the isotonic agent is about 0.1-1%. In certain embodiments, the concentration of the isotonic agent is about 0.5-3%. In certain embodiments, the concentration of the isotonic agent is about 0.25 wt %. In certain embodiments, the concentration of the isotonic agent is about 0.45 wt %. In certain embodiments, the concentration of the isotonic agent is about 0.9 wt %. In certain embodiments, the concentration of the isotonic agent is about 1.2 wt %. In certain embodiments, the concentration of the isotonic agent is about 2.4 wt %. In certain embodiments, the concentration of the isotonic agent is about 5 wt %.

In some embodiments, isotonic agents may be present in compositions and/or formulations in one or more of the aforementioned ranges during the forming and/or dilution processes described herein. In certain embodiments, isotonic agents may be present in compositions and/or formulations in one or more of the aforementioned ranges in the final product.

Polydispersity is a measure of the non-uniformity of particle size in a formulation. Non-uniformity in particle size may be due to differences in individual particle size and/or the presence of agglomerates in the formulation. A formulation comprising the particles is considered substantially homogeneous or “monodisperse” if the particles have essentially the same size, shape, and/or mass. Formulations comprising particles of various sizes, shapes, and/or masses are considered to be heterogeneous or “polydisperse”.

In some embodiments, in the presence of added ionic strength and/or when the added ionic strength of the composition and/or formulation remains relatively constant or increases (eg, during the formation and/or dilution process) the composition and/or or the polydispersity of the formulation is relatively constant. In certain embodiments, when the ionic strength increases by at least 50%, the polydispersity is about 200% or less, about 150% or less, about 100% or less, about 75% or less, about 50% or less, about 30% or less, about 20% or less, about 10% or less, about 3% or less, or about 1% or less. In certain embodiments, when the ionic strength is increased by at least 50%, the polydispersity increases by at least about 1%, at least about 3%, at least about 10%, at least about 30%, or at least about 100%. Combinations of the above-mentioned ranges are possible (eg, an increase in polydispersity of 1% or less of 50% or less). Other ranges are also possible.

The ionic strength of the formulations described herein can be controlled (eg, increased) by various means, such as adding one or more ionic isotonic agents (eg, salts, such as NaCl) to the formulation. In certain embodiments, the ionic strength of the formulations described herein is at least about 0.0005 M, at least about 0.001 M, at least about 0.003 M, at least about 0.01 M, at least about 0.03 M, at least about 0.1 M, at least about 0.3 M, at least about 1 M or greater, about 3 M or greater, or about 10 M or greater. In certain embodiments, the ionic strength of the formulations described herein is about 10 M or less, about 3 M or less, about 1 M or less, about 0.3 M or less, about 0.1 M or less, about 0.03 M or less, about 0.01 M or less, about 0.003 M or less. M or less, about 0.001 M or less, or about 0.0005 M or less. Combinations of the aforementioned ranges are possible (eg, ionic strengths greater than or equal to about 0.01 M and less than or equal to about 1 M). Other ranges are also possible. In certain embodiments, the formulations described herein have an ionic strength of about 0.1 M. In certain embodiments, the formulations described herein have an ionic strength of about 0.15 M. In certain embodiments, the formulations described herein have an ionic strength of about 0.3 M.

The polydispersity of the formulations described herein can be measured by the polydispersity index (PDI). PDI is used to describe the width of a particle size distribution and is often calculated from cumulants analysis of dynamic light scattering (DLS) measured intensity autocorrelation functions. The calculation of these parameters is specified in the standards ISO 13321: 1996 E and ISO 22412: 2008. PDI is dimensionless and, as measured by DLS, values below 0.05 indicate highly monodisperse samples, while values above 0.7 are normalized to indicate a very broad size distribution. In certain embodiments, the PDI of the formulations and/or compositions described herein is about 1 or less, about 0.9 or less, about 0.8 or less, about 0.7 or less, about 0.6 or less, about 0.5 or less, about 0.4 or less, about 0.3 or less, about 0.2 or less. or less, about 0.15 or less, about 0.1 or less, about 0.05 or less, about 0.01 or less, or about 0.005 or less. In certain embodiments, the PDI of the formulations and/or compositions described herein is at least about 0.005, at least about 0.01, at least about 0.05, at least about 0.1, at least about 0.15, at least about 0.2, at least about 0.3, at least about 0.4, at least about 0.5 or greater, about 0.6 or greater, about 0.7 or greater, about 0.8 or greater, about 0.9 or greater, or about 1 or greater. Combinations of the aforementioned ranges are possible (eg, PDI of greater than or equal to about 0.1 and less than or equal to about 0.5). Other ranges are also possible. In certain embodiments, the PDI of the formulation is about 0.1. In certain embodiments, the PDI of the formulation is about 0.15. In certain embodiments, the PDI of the formulation is about 0.2.

In certain embodiments, the compositions and/or formulations described herein may be highly dispersible and do not tend to form agglomerates. Even when the particles form agglomerates, the agglomerates can be readily broken down into individual particles without rigorous agitation of the composition and/or formulation.

The compositions and/or formulations described herein may have any suitable pH value. The term “pH”, unless otherwise provided, refers to the pH measured at ambient temperature (eg, about 20° C., about 23° C., or about 25° C.). A composition and/or agent has, for example, an acidic pH, a neutral pH, or a basic pH, which may be determined by, for example, where the composition and/or agent is delivered into the body. In certain embodiments, the composition and/or formulation has a physiological pH. In certain embodiments, the pH value of the composition and/or formulation is at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 6.2, at least about 6.4, at least about 6.6, About 6.8 or more, about 7 or more, about 7.2 or more, about 7.4 or more, about 7.6 or more, about 7.8 or more, about 8 or more, about 8.2 or more, about 8.4 or more, about 8.6 or more, about 8.8 or more, about 9 or more, about 10 or more or more, about 11 or more, or about 12 or more. In certain embodiments, the pH value of the composition and/or formulation is about 12 or less, about 11 or less, about 10 or less, about 9 or less, about 8.8 or less, about 8.6 or less, about 8.4 or less, about 8.2 or less, about 8 or less; about 7.8 or less, about 7.6 or less, about 7.4 or less, about 7.2 or less, about 7 or less, about 6.8 or less, about 6.6 or less, about 6.4 or less, about 6.2 or less, about 6 or less, about 5 or less, about 4 or less, about 3 or less or less, about 2 or less, or about 1 or less. Combinations of the aforementioned ranges are possible (eg, a pH value of at least about 5 and up to about 8.2). Other ranges are also possible. In certain embodiments, the pH value of the compositions and/or formulations described herein is greater than or equal to about 5 and less than or equal to about 8.

In one set of embodiments, a composition comprising a plurality of coated particles is provided. The coated particles comprise core particles comprising or formed from a solid drug or a salt thereof, wherein the drug or salt is no more than about 1 mg/mL (e.g., 25 °C) at 25 °C at any point throughout the pH range. has a solubility in water of about 0.1 mg/mL or less in make up The coated particles also comprise a coating comprising a surface modifier surrounding the core particle, wherein the surface modifier comprises a triblock copolymer comprising a hydrophilic block-hydrophobic block-hydrophilic block configuration, wherein the hydrophobic block has a molecular weight of at least about 3 kDa, and the hydrophilic block constitutes at least about 30 wt % of the triblock, wherein the hydrophilic block is or comprises poly(ethylene oxide) having a molecular weight of at least about 2 kDa, wherein the hydrophobic block comprises: Associated with the surface of the core particle (eg, by adsorption), wherein the hydrophilic block is present at the surface of the coated particle to render the coated particle hydrophilic, wherein the surface-altering agent is about 0.001 molecules per nanometer square present on the surface of the core particle at a density of greater than or equal to (eg, greater than about 0.01 molecules per nanometer squared). The coated particles have a relative velocity greater than 0.5 in mucus. The coated particles may have an average size of about 20 nm or more and about 1 μm or less (eg, about 500 nm or less). The thickness of the coating can be, for example, about 50 nm or less (eg, about 30 nm or less, about 10 nm or less). The composition may be a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, additives and/or diluents. In some embodiments, the plurality of coated particles comprises one or more glass surface modifying agents in the composition (e.g., Pluronic® P123, Pluronic® P103, Pluronic® P105, Pluronic® F127, Flu poloxamer selected from Ronic® F108 and combinations thereof) in solution (eg, aqueous solution). The glass surface modifying agent(s) in solution and the surface modifying agent on the surface of the particle may be the same surface modifying agent(s) and may be in equilibrium with each other in the composition. The total amount of surface-altering agent present in the composition may be, for example, from about 0.001% to about 5% by weight (eg, from about 0.01% to about 5%, or from about 0.1% to about 5%) by weight. can In some embodiments, the composition has a PDI of about 0.1 or greater and about 0.5 or less (eg, about 0.3 or less, or about 0.2 or less). Methods of use and/or delivery of such compositions to a patient or subject (eg, to mucus or mucous membranes) are also provided.

In one set of embodiments, a composition comprising a plurality of coated particles is provided. The coated particles comprise core particles comprising or formed from a solid drug or a salt thereof, wherein the drug or salt is no more than about 1 mg/mL (e.g., 25 °C) at 25 °C at any point throughout the pH range. has a solubility in water of about 0.1 mg/mL or less in make up The coated particles also include a coating comprising a surface-altering agent surrounding the core particle, wherein the surface-altering agent is a poloxamer comprising a hydrophilic block-hydrophobic block-hydrophilic block configuration, wherein the poloxamer is pluronic ® P123, Pluronic® P103, Pluronic® P105, Pluronic® F127, Pluronic® F108 and combinations thereof, wherein the hydrophobic block associates with the surface of the core particle (e.g., by adsorption), wherein the hydrophilic block is present on the surface of the coated particle to render the coated particle hydrophilic, wherein the surface-altering agent is at least about 0.001 molecules per nanometer square (e.g., about 0.01 per nanometer square). more than molecules) on the surface of the core particles. The coated particles have a relative velocity greater than 0.5 in mucus. The cored particles can have an average size of about 20 nm or more and about 1 μm or less (eg, about 500 nm or less). The thickness of the coating can be, for example, about 50 nm or less (eg, about 30 nm or less, about 10 nm or less). The composition may be a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, additives and/or diluents. In some embodiments, the plurality of coated particles comprises one or more free poloxamers in the composition (eg, Pluronic® P123, Pluronic® P103, Pluronic® P105, Pluronic® F127, Pluronic). nick® F108 and combinations thereof) in solution (eg, aqueous solution). The free poloxamer(s) in solution and the poloxamer on the surface of the particle may be the same poloxamer(s) and may be in equilibrium with each other in the composition. The total amount of poloxamer present in the composition can be, for example, from about 0.001% to about 5% by weight (e.g., from about 0.01% to about 5% by weight, or from about 0.1% to about 5% by weight). have. In some embodiments, the composition has a PDI of about 0.1 or greater and about 0.5 or less (eg, about 0.3 or less, or about 0.2 or less). Methods of use and/or delivery of such compositions to a patient or subject (eg, to mucus or mucous membranes) are also provided.

In some of the embodiments described above, the poloxamer is selected from Pluronic® P123, Pluronic® P103, Pluronic® P105, Pluronic® F127, and combinations thereof. In some embodiments, the poloxamer is not Pluronic® F68 or Pluronic® F108.

These and other aspects of the invention will be further appreciated upon consideration of the following examples, which are intended to illustrate certain specific embodiments of the invention, but to limit the scope thereof, as defined by the claims. It's not what you want to do.

Example

Example 1

The following describes non-limiting examples of methods for forming non-polymeric solid particles into mucus-penetrating particles. Pyrene, a hydrophobic, naturally fluorescent compound, was used as the core particle and prepared by a nanomilling process in the presence of various surface modifiers. The stabilizer acted as a surface modifier and formed a coating around the core particle. Different stabilizers/surface modifiers were evaluated to determine the effectiveness of the coated particles in penetrating mucus.

Nanomilling of pyrene in aqueous dispersions in the presence of various stabilizers/surface modifiers to determine whether certain stabilizers/surface modifiers can 1) aid in particle size reduction to hundreds of nanometers and 2) particles with mucus constituents. It was determined whether it was possible to physically (non-covalently) coat the surface of the resulting nanoparticles with a mucoinert coating that would minimize interaction and prevent mucus adhesion. In these experiments, the stabilizer/surface-altering agent acted as a coating around the core particles and the resulting particles were tested for their mobility in mucus, but in other embodiments, the stabilizer/surface-altering agent was applied to the particles in the mucus. It can be replaced with other surface modifiers that can increase mobility. Stabilizers/surface modifiers tested include pharmaceutically relevant excipients such as poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymers (Pluronics ^® ), polyvinylpyrrolidone ( various other polymers, oligomers and small molecules listed in Table 2, including kollidone) and hydroxypropyl methylcellulose (Methocel), and the like.

An aqueous dispersion containing pyrene and one of the stabilizers/surface modifiers listed above was milled with milling media until the particle size was reduced to less than 500 nm. Table 3 lists the particle size characteristics of the pyrene particles obtained by nanomilling in the presence of various stabilizers/surface modifiers. The particle size was determined by dynamic light scattering. When Pluronics ^® L101, L81, L44, L31, Span 20, Span 80, or octyl glucoside were used as stabilizers/surface modifiers, stable nanosuspensions could not be obtained. Therefore, these stabilizers/surface modifiers were excluded from further investigation due to their inability to effectively aid in particle size reduction.

Mobility and distribution of pyrene nanoparticles from nanosuspensions prepared in human cervical mucus (CVM) were characterized using fluorescence microscopy and multi-particle tracking software. In a typical experiment, ≤0.5 uL of nanosuspension (diluted if necessary to a surfactant concentration of ˜1%) along with control was added to 20 μl of fresh CVM. Conventional nanoparticles (200 nm yellow-green fluorescent carboxylate-modified polystyrene microspheres, obtained from Invitrogen) were used as negative controls to confirm the blocking properties of CVM samples. Red fluorescent polystyrene nanoparticles covalently coated with PEG 5 kDa were used as positive controls with an established MPP behavior. Using a fluorescence microscope equipped with a CCD camera, each type of particle: sample (pyrene), negative control, and positive control (natural blue fluorescence of pyrene, which allowed observation of pyrene nanoparticles separately from control) A 15 s movie was captured at a temporal resolution of 66.7 ms (15 frames/s) under 100x magnification from several regions within each sample for Then, using advanced image processing software, the individual trajectories of multiple particles were measured over a time scale of 3.335 s (50 frames) or longer. The resulting transport data are presented here in the form of an orbital-averaged velocity V _mean , i.e., the velocity of an individual particle averaged over its orbit, and an ensemble-averaged velocity <V _{mean >, i.e., V mean} averaged over an ensemble of particles. presented. To enable easy comparison between different samples and to normalize the rate data for the natural variability of penetrability of CVM samples, the relative sample rate <V _mean > _relative was determined according to the equation shown in Equation 1.

Before quantifying the mobility of the prepared nanoparticles, their spatial distribution in mucus samples was evaluated by microscopy at low magnifications (10x, 40x). The pyrene/methocell nanosuspension did not achieve a uniform distribution among the CVMs and was found to be strongly aggregated into domains much larger than the mucus mesh size (data not shown). Such aggregation is indicative of mucoadhesive behavior and effectively prevents mucus penetration. Therefore, further quantitative analysis of particle mobility was deemed unnecessary. Similar to the positive control, all other tested pyrene/stabilizer systems achieved a fairly uniform distribution among CVMs. Multi-particle tracking showed that the negative control was highly constrained in all tested samples, while the positive control was highly mobile, as evidenced by _{the <V mean} > for the positive control being significantly greater than that for the negative control. was confirmed (Table 4).

Nanoparticles obtained in the presence of certain (but important, but not all) stabilizers/surface-altering agents were found to migrate through the CVM at the same or nearly the same rate as the positive control. Specifically, Pluronics ^® F127, F108, P123, P105, and P103 stabilized pyrene nanoparticles, as shown in Table 4 and Figure 2A, _{exceeded the <V mean} > of the negative control by approximately 10-fold, _{The <V mean} > indistinguishable from that of the positive control group was shown within the error. For these samples, the <V _mean > _relative values were greater than 0.5, as shown in FIG. 2B .

On the other hand, the pyrene nanoparticles obtained using other stabilizers/surface modifiers were mostly or completely passivated, as evidenced by _{their respective <V mean} > _{relative values of 0.4 or less, and most of the stabilizers/surface} When the modifier was used, it was less than or equal to 0.1 (Table 4 and Figure 2B). Furthermore, Figures 3A-3D are histograms showing the distribution of _{V-means within an ensemble of particles.} These histograms show the ^{mucus of samples stabilized with Pluronic ®} F127 and Pluronic ^® F108 in ^{contrast to the mucoadhesive behavior of samples stabilized with Pluronic ®} 87 and Kollidon 25 (selected as representative mucoadhesive samples). - muco-diffusive behavior ( ^{similar histograms were obtained for samples stabilized with Pluronic ®} P123, P105, and P103, but not shown here) are illustrated.

To determine the characteristics of ^{Pluronics ®} that make pyrene nanocrystals mucus penetrating, the <V _average > _relative ^{of pyrene/pluronic ®} nanocrystals was used to determine the molecular weight and PEO weight content (%) of the PPO block of ^{Pluronics ® used.} ) was mapped to ( FIG. 4 ). ^{It was concluded that such Pluronics ®} having at least a PPO block of at least 3 kDa and a PEO content of at least about 30 wt % made the nanocrystals mucus-penetrating. Without wishing to be bound by any theory, a hydrophobic PPO block may provide effective association with the surface of the core particle provided that the molecular weight of the block is sufficient (eg, at least about 3 kDa in some embodiments); On the other hand, the hydrophilic PEO block is present at the surface of the coated particle and shields the coated particle from adhesion interactions with the mucin fiber when the PEO content ^{of Pluronic ® is sufficient (eg, at least 30 wt % in some embodiments).} It is believed that it can be done As described herein, in some embodiments the PEO content of the surface modifier is at least about 10 wt% (e.g., at least about 15 wt%, or about 20 wt% or more).

Example 2

This example describes the formation of mucus-penetrating particles using various non-polymeric solid particles.

The technique described in Example 1 was applied to other non-polymeric solid particles to confirm the versatility of the approach. F127 was used as a surface modifier to coat various active agents used as core particles. Sodium dodecyl sulfate (SDS) was selected as a negative control to compare each drug with similarly sized nanoparticles of the same compound. An aqueous dispersion containing drug and Pluronic® F127 or SDS was milled with milling media until the particle size was reduced to less than 300 nm. Table 5 lists the particle sizes for a representative selection of drugs milled using this method.

To measure the ability of drug nanoparticles to penetrate mucus, a new assay was developed to measure the bulk transport of nanoparticles into mucus samples. Most drugs are not naturally fluorescent and therefore difficult to measure with particle tracking microscopy techniques. The newly developed bulk transport assay does not require that the analyzed particles be fluorescent or labeled with a dye. In this method, 20 μL of CVM was collected in a capillary and sealed at one end with clay. The open end of the capillary was then immersed in 20 μL of an aqueous suspension of particles (which is 0.5% w/v drug). After the desired time, typically 18 hours, the capillary tube was removed from the suspension and the exterior was wiped clean. The capillary containing the mucus sample was placed in an ultracentrifuge tube. The extraction medium was added to the tube and incubated for 1 hour with mixing, which was to remove the mucus from the capillary and extract the drug from the mucus. The samples were then spun to remove mucins and other insoluble components. The amount of drug in the extracted samples could then be quantified using HPLC. The results of these experiments are very consistent with those of microscopy, indicating a clear differentiation in transport between mucus penetrating particles and conventional particles (CP). The transport results for a representative selection of drugs are shown in FIG. 5 . These results corroborate the findings of microscopy/particle tracking with pyrene and extend to common active pharmaceutical compounds; We demonstrate that coating non-polymeric solid nanoparticles with F127 enhances mucus penetration.

In Examples 1-2, cervical mucus (CVM) samples were obtained from healthy female volunteers 18 years of age or older. CVM was collected by inserting a Softcup® menstrual collection cup into the vaginal tract as described by the product literature for 30 seconds to 2 minutes. After removal, CVM was then collected from Softcup® by gentle centrifugation at ˜30×G to ˜120×G in a 50 mL centrifuge tube. In Example 1, CVM was used undiluted and fresh (stored for up to 7 days under refrigerated conditions). Barrier and transport of all CVM samples used in Example 1 were demonstrated with negative (200 nm carboxylated polystyrene particles) and positive (200 nm polystyrene particles modified with PEG 5K) controls. In Example 2, CVM was lyophilized and reconstituted. In Example 2, the mucus was frozen at -50°C and then lyophilized to dryness. The samples were then stored at -50°C. Prior to use, the solid was ground into a fine powder using a mortar and pestle, and then water was added to restore the mucus from the original volume to a final volume equivalent to twice the original volume. The reconstituted mucus was then incubated at 4° C. for 12 hours and used as described in Example 2. Barrier and transport of all CVM samples used in Example 2 were demonstrated with negative (200 nm carboxylated polystyrene particles) and positive (F127 coated 200 nm polystyrene particles) controls.

Example 3

This example describes the formation of mucus-penetrating particles using a core comprising the drug loteprednol etabonate (LE).

To demonstrate the value of enhanced mucus penetration in the delivery of non-polymeric solid particles, an MPP formulation of loteprednol etabonate (LE MPP; LE particles coated with Pluronic® F127 prepared by the method described in Example 2) ) was compared with the currently marketed formulation, Rotemax®. Rotemax® is a steroid eye drop approved for the treatment of ocular surface inflammation. Conventional particles, such as those in Rotemax®, are widely captured by the surrounding rapidly cleared mucus layer in the eye and are thus also rapidly cleared. LE MPP avoids adhesion to mucus and can penetrate mucus effectively, facilitating sustained drug release directly into the underlying tissue. Improving drug exposure at the target site will allow the overall dose to be reduced, which increases patient compliance and safety. In vivo, a single topical instillation of LE MPP in New Zealand white rabbits resulted in significantly higher drug levels in the blepharic conjunctiva, ocular conjunctiva, and cornea compared to an equivalent dose of ^{Rotemax ® ( FIGS. 6A-6C )} . LE levels from MPP at 2 hours were 6, 3, and 8 fold higher than from Rotemax® (blepharo, ocular, and cornea, respectively). In particular, the LE level from the MPP is approximately two times higher at 2 hours than the level from Rotemax® at 30 minutes. These results demonstrate the utility of the non-polymeric solid MPP approach.

Example 4

This example describes the formation of mucus-penetrating particles comprising a core comprising curcumin (CUR).

Molecules with varying solubility were selected as model therapeutics to form particles with the core of a solid drug. One of them, curcumin, is proposed to have antioxidant, anti-tumor and anti-inflammatory properties. It is an interesting candidate not only for its wide potential medical applications, but also because of its high hydrophobicity and natural fluorescence. The former properties mean that CUR is poorly soluble in aqueous solutions, while the latter allows for rapid and label-free detection and characterization of particles. The particles were coated with a surfactant (eg, Pluronic ^® F127, abbreviated as F127 in Examples 4 and 5) to render the particles mucus penetrating.

A simple procedure based on sonication was developed to formulate particles of CUR. Briefly, 5 mg CUR was dispersed in a 2 mL aqueous solution containing F127 (or other surfactant) in a 7 mL scintillation vial. The suspension was sonicated in a water bath for 20 minutes. The curcumin suspension was then sonicated using a sonicator equipped with a 3 mm stepped probe at 100% amplitude for 30 minutes. The suspension was centrifuged at 2000 rpm for 10 minutes to remove undestroyed crystals. The supernatant was stored at 4C for 2 hours. The supernatant was centrifuged at 16,500 rpm for 20 min, then the pellet was collected. It was noted that, without sufficient incubation time prior to particle collection, the diffusivity of the coated particles was much lower (data not shown), suggesting the importance of the dense F127 coating in the production of mucus-penetrating particles.

Table 6 and (Figures 7A-7B) summarize the physicochemical properties of the resulting coated CUR particles prepared using the above method. CUR particles formulated at 1% (w/v) F127 (CUR-1% F127 particles) had an average size of 133 nm, which was consistent with observations through TEM images ( FIG. 7B ). The zeta-potential was close to neutral. As the concentration of F127 in the CUR suspension was decreased during sonication, the CUR particle size and polydispersity (PDI) respectively increased, presumably as a result of the decreased F127 coating density resulting in a weakened stabilizing effect on the CUR particles (Table 6). The F127 concentration had little effect on the particle zeta-potential, probably due to the deionization of curcumin at pH 4. Powder-XRD measurements on CUR-1% F127 particles showed that the chemical structure and crystallinity of CUR was not altered by either sonication or incorporation of F127 ( FIG. 7A ).

To investigate the mucus-penetrating properties of CUR-F127 particles, transport of CUR-1% F127 particles was performed using multi-particle tracking (MPT) in both human cervicologic mucus (CVM) and human cystic fibrosis sputum (CFS) samples. was used to study. Briefly, particles were added to mucus samples and their migration was recorded using high resolution epi-fluorescence microscopy. Then, their trajectories and transport rates were analyzed and quantified. 8A-8B show the time-dependent ensemble averaged geometric mean square displacement (<MSD>) of CUR-1% F127 particles in both CVM and CFS. 200 nm PEGylated (PEG) and carboxylated (COOH) polystyrene (PS) particles were selected as muco-inert and muco-adhesive controls, respectively. For the entire range of time scales studied, the <MSD> of CUR-1% F127 particles was comparable to that of PSPEG in CVM and significantly higher than that of PSCOOH in both types of mucus samples. At a time scale of 1 s, the <MSD> of CUR-1% F127 particles was 4400-fold and 220-fold higher than that of PSCOOH in CVM and CFS, respectively. The geometric ensemble averaged effective diffusivity (<Deff>) of CUR-1% F127 particles at a time scale of 1 s was only 9 times lower than the calculated theoretical diffusivity in water (Table 6).

To further investigate the effect of the coating density of F127 on the transport of CUR particles in mucus, the particles were formulated at various F127 concentrations and their diffusivity was characterized in human CVM ( FIG. 9 , Table 6). The <Deff> of CUR-0.1% and 0.01% F127 particles was similar or slightly reduced compared to that of CUR-1% F127 particles, but transport of CUR-0.001% F127 particles was dramatically retarded ( FIG. 9 ). The diffusivity of CUR-0.001% F127 particles was 10000 times slower in human CVM compared to in water, and about 1000 times lower than that of CUR-1% F127 particles in CVM. In general, decreasing the F127 concentration in the preparation of CUR particles decreased the diffusivity, presumably because the reduced F127 concentration lowered the surface density of F127 at equilibrium (thus brushing the PEG). It was not evident that this effect would be so pronounced when the F127 concentration was lowered to less than 0.01% (w/v).

In addition to F127, various Pluronics ^® have been used in pharmaceutical applications. Different Pluronics ^® were tested to see if they would work equally well, or if only certain would convert CUR particles into mucus-penetrating particles. ^{Twelve additional Pluronics ®} as listed in Table 7 (in increasing order of PPO MW) were selected and the corresponding CUR particles were prepared. The resulting sizes ranged from 90 to 232 nm, with most staying in the 100-150 nm range. All particle types showed a PDI greater than that of the CUR-1% F127 particles (0.33), mostly falling between 0.4-0.6. These results meant that F127 could have the strongest stabilizing effect among all tested Pluronics ^®. The zeta-potential was uniformly neutral for all studied CUR particles.

The transport rates of CUR particles coated with various Pluronics ^® were characterized in human CVM (Table 7). By comparing their diffusivity to that of CUR-1% F127 particles (Dw/Dm = 9), they were grouped into three groups: particle migration strongly retarded (F65, F68; Dw/Dm > 100), particle migration retarded. (F38, F84, F85, F88; 20 ≤ Dw/Dm ≤ 100), and particles penetrate rapidly (F98, P103, P104, P105, F108, F123; Dw/Dm < 20). The fact that all of the particles had a nearly neutral surface charge, but only the ^{formulations with certain Pluronic ®} coatings (eg F65 and F68) showed strongly retarded diffusivity, the adhesion between these CUR particles and the mucus component suggests that sex is likely governed by hydrophobic interactions,

To identify particles that determine the diffusivity of CUR particles coated with different Pluronics ^® , the <Deff> of CUR particles (at a time scale of 1 s) was mapped against the PPO and PEG MW of ^{Pluronics ® used (} Figure 10A). A general increase in <Deff> was observed with the growth length of the PPO segment (Y-axis), but there was still no specific pattern between <Deff> and PEG MW (X-axis). Surprisingly, this transition occurred at a PPO MW of approximately 2000 Da, which is smaller than previously found for ^{Pluronic ® coated polystyrene particles.} Particle diffusivity showed a strong correlation with PPO MW (R=0.92), but little correlation with PEG MW (R=0.14). ^{It is possible that Pluronics ®} with longer PPO segments has a higher affinity to the hydrophobic surface of the CUR particles, thus anchoring more tightly and providing a denser and more stable shielding on the surface.

^{To explore the ability of surfactants other than Pluronics ®} to potentially generate mucus-penetrating particles, Tween 20, Tween 80 and Vitamin E TPGS, all containing a PEG segment, were tested. The characteristics of the CUR particles prepared in these surfactants are shown in Table 8. Although all three groups exhibited a particle size of approximately 150 nm and a near-neutral zeta-potential, their diffusivity was greatly reduced in human CVM compared to that in water. The lower transport rate may indicate an ineffective coating of surfactant molecules on the surface of the CUR particles, which may be attributed to their shorter hydrophobic segment compared to F127.

To evaluate the ability of CUR particles to deliver CUR in a sustained manner, the release profile of CUR-1% F127 particles was characterized. Briefly, dissolved CUR was extracted by suspending a known amount of CUR particles in phosphate buffered saline (PBS, pH 7.4) in a 50 mL test tube with a layer of octanol on top. The suspension was incubated at 37°C with stirring. Octanol was collected and replaced at each time point. The concentration of CUR in octanol was determined fluorometrically. 11 , the CUR-1% F127 particles provided continuous release for 24-48 hours in vitro. About 80% of the CUR content was released within the first 24 hours.

Example 5

The development of mucus-penetrating particles is described using the hydrophobic drug, 5,10,15,20-tetra(p-hydroxyphenyl)porphyrin (p-THPP).

Besides CUR, the same method described in Example 4 was applied to the hydrophobic drug p-THPP. p-THPP is a therapeutic agent used in photodynamic therapy to treat cancer and has been selected as a model photosensitizer in previous studies. The basic properties of p-THPP-1% F127 particles, including size, zeta-potential and diffusivity in human CVM, were determined according to the previously described procedure (Table 9). Similar to the CUR-1% F127 particles, the p-THPP-1% F127 particles exhibited a size of 187 nm and a near-neutral surface charge. The diffusivity of p-THPP-1% F127 particles in human CVM was only 8 times slower than that in water, indicating that the particles were not immobilized by the adherent component in the mucus and at a rate comparable to that of CUR particles-1% F127 mucus. It indicates that it is capable of diffusing through the gel.

Example 6

This example describes the development of mucus-penetrating particles using tenofovir (TFV) and acyclovir monophosphate (ACVp), highly water-soluble drugs.

Tenofovir (TFV) is a potent antiviral drug used to treat infectious diseases. Due to the fact that tenofovir (TFV) is highly water-soluble, a method has been developed for formulating mucus-penetrating particles of tenofovir. The water solubility of TFV is greater than 15 mg/mL, so conventional techniques for the preparation of insoluble particles or, alternatively, encapsulation with hydrophobic polymer nanoparticles have not been successful. To reduce the water solubility of TFV, the interaction between cations and nucleotide/nucleotide analogs was exploited. TFV interacts very strongly with zinc cations (Zn) through phosphonate groups and purine ring structures. This interaction with zinc causes TFV precipitation into crystals that can be stabilized by the coatings described herein to stop agglomeration and determine crystal surface properties. Furthermore, Zn is naturally present in vaginal fluid and has known antibacterial properties and has recently been extended to include anti-HIV activity.

It is essential to ensure that the TFV-Zn particles will exhibit extended release into the buffer, since the crystals and coatings are formed entirely by non-covalent interactions. When compared to the solution of free TFV, the particles exhibited a much slower release from the 100 kDa dialysis membrane (ca. 40% over 24 h) ( FIG. 12 ).

The TFV-Zn particles were then formulated with either F127 or PVA coatings. As shown in Table 10, the presence of the coating stabilized the particles, as evidenced by a smaller average size and reduced polydispersity. The presence of a coating on the surface also suggests a shift in the zeta potential towards a more neutral charge. Furthermore, these particles were fluorescently labeled for imaging purposes, via covalent attachment of Alexa Fluor® dye to free amines on TFV. The crystals were found to be unstable when prepared at a ratio of 1:50 labeled:unlabeled TFV, but were visible and stable through fluorescence microscopy at a ratio of 1:200 labeled:unlabeled TFV.

After obtaining stable, fluorescently labeled particles, it was determined whether the F127 coating improved particle distribution at the mucosal surface in animals, as consistent with the use of coated polymer nanoparticles. TFV particles coated with either F127 or PVA were administered to the vagina of mice, then mice were sacrificed, their vaginas dissected, flattened on microscope slides, and imaged. As shown in FIGS. 13A-13B , F127-coated particles were well distributed over the entire vaginal surface, while PVA-coated particles had no indication that they did not enter the vaginal folds (rugae). It exhibited incomplete coverage of the vaginal surface, evident as a "striping" behavior.

MPP particles were then prepared using acyclovir monophosphate (ACVp) for use in testing the potential efficacy of MPP particles in preventing infection by herpes simplex virus type 2 (HSV-2). ACVp is not dependent on viral thymidine kinase for an early phosphorylation step that can lead to viral resistance. It also provides ACVp anti-HIV activity, albeit with low potency.

6-8 week old female CF-1 mice were injected subcutaneously with medroxyprogesterone acetate, with fire-polished positive displacement capillary pipettes (Wiretrol, Drummond). Scientific)), and 20 μL of test agent or PBS was given intravaginally after 1 week. After 30 minutes, mice were challenged with 10 uL of inoculum containing HSV-2 strain G (ATCC #VR-734, 2.8 x 10 ⁷ TCID _{50 per mL).} HSV-2 was diluted 10-fold with Bartel's medium to deliver 10 ID ₅₀ (a dose that typically infects ˜85% of control mice). As previously described (RA Cone, T. Hoen, X. Wong, R. Abusuwwa, DJ Anderson, TR Moench, Vaginal microbicides: detecting toxicities in vivo that paradoxically increase pathogen transmission. BMC Infect Dis 6, 90 (2006) )]), mice were evaluated for infection 3 days after inoculation by culturing PBS vaginal washes on human foreskin fibroblasts (Diagnostic Hybrids, MRHF Lot #440318W). In this model, the input (challenge) virus was no longer detectable in the lavage fluid when collected more than 12 hours after challenge.

It has been found that ACVp particles can be prepared in the presence of zinc, via interactions similar to those in TFV (phosphate groups and purine rings). Soluble ACVp or ACVp in the form of MPP nanocrystals was administered to mice 30 min prior to challenge with live HSV. Both drug and virus were administered intravaginally. Soluble drug administered at the same concentration as the MPP-drug was ineffective (84% infected compared to 88% of the control group), while only 46.7% of mice in the MPP-drug group were infected. Groups of mice given soluble drug at 10 times the dose given MPP-drug were infected at a rate of 62% (drug in PBS) or 69.3% (drug in water). When soluble drug was compared to MPP-drug administered in the same vehicle (pure water), soluble drug was less protective than MPP-drug even at 10-fold higher concentrations (p=0.02).

Note that stable nanocrystals of drugs with free phosphate groups can be formed using Zn using both freeze-drying approaches and sonication procedures. For freeze-drying, the drug was dissolved in an aqueous solution of F127. Zinc acetate was added in the range of 1:50 to 1:5 (Zn: drug), and the solution was immediately flash frozen. The dried powder was reconstituted in water at the desired concentration. F127 concentrations both above (1%) and below (0.08%) the critical micelle concentration (CMC) of F127 can be used to prepare stable TFV nanocrystals. However, ACVp nanocrystals were much more sensitive to surfactant concentration. Stable nanocrystals could only be formed when using a F127 concentration below the CMC (~0.1%). F127 concentrations above CMC caused significant aggregation and sedimentation of ACVp nanocrystals, regardless of the formulation method tested.

Furthermore, stable nanocrystals can be formed by adding excess zinc acetate to the drug solution. The precipitate was washed 3+ times by centrifugation. The resulting slurry was sonicated using a probe sonicator (without surfactant; bubbling causes agglomeration and instability). Other methods, such as milling, may also work. A surfactant was then added to stabilize the resulting nanocrystals. Without the presence of surfactant, significant agglomeration and sedimentation occur. Using this formulation technique, stable TFV nanocrystals could be prepared using, for example, F127 concentrations of 1% or less. Similarly, in these experiments, stable ACVp nanocrystals could be formed using only F127 concentrations below CMC (typically 0.08% used).

In laboratories 4-6, normal vaginal flora was harvested using a self-sampling menstrual collection device according to a protocol approved by the Institutional Review Board of the Johns Hopkins University. undiluted cervical secretions were collected from women with The device was inserted into the vagina for -30 s, removed, and placed in a 50 mL centrifuge tube. Samples were centrifuged at 1,000 rpm for 2 minutes to collect mucus secretions.

In Examples 4-6, a 100x oil-immersion objective (NA, 1.46) was mounted on an inverted epifluorescence microscope (Chice, Thornwood, NY) equipped with an appropriate filter. By analyzing the trajectories of fluorescently or fluorescently labeled particles, recorded using an electron multiplying charge coupled device (EMCD) camera (Evolve 512, Photometrics, Tucson, Arizona). The particle transport rate was measured. Experiments were performed on custom-made chamber slides, where a diluted particle solution (0.0082% wt/vol) was added to 20 µL of fresh mucus at a concentration of 3% v/v (final particle concentration, 8.25 x 10 ^-7 wt/vol). It was added to final concentration and stabilized at room temperature prior to microscopy. Orbitals of n ≥ 100 particles were analyzed for each experiment, and at least 3 independent experiments were performed for each condition. Movies were captured using Metamorph software (Universal Imaging, Glendale, Wis.) at a temporal resolution of 66.7 ms for 20 s. The tracking resolution was 10 nm, as determined by tracking the displacement of particles immobilized with strong adhesion. The coordination of the nanoparticle centroid was converted to a time-averaged MSD, which is <Δr2(τ)> = [x(t+τ) - x(t)] ² + [y(t+τ) - y(t)] ² (where x and y represent the nanoparticle coordination at a given time and τ is the time scale or time lag). The distribution of MSD and effective diffusivity was calculated from this data. Particle penetration into the mucus layer was modeled using the diffusion coefficients obtained from Pick's second law and tracking experiments.

Example 7

This example describes the development of mucus-penetrating particles to improve drug delivery to the mucosal surface of the mouse vagina.

Improved methods for sustained and more uniform drug delivery to the vagina could provide more effective prevention and treatment of conditions affecting women's health, such as cervical cancer, bacterial vaginosis, and sexually transmitted infections. have. For example, women become disproportionately infected with HIV, in part due to the lack of woman-controlled prophylaxis. An easily administered, prudent, and effective method for protecting women against vaginal HIV transmission could prevent millions of infections worldwide. However, vaginal folds, or “mucosal folds,” which seek to expand during sexual intercourse and childbirth, typically collapse by intraperitoneal pressure, making the surface of these folds less accessible to the drug and drug carrier. Poor distribution into the vaginal folds even after simulated intercourse has been cited as a critical factor in failing to protect the susceptible vaginal surface from infection. Distribution over the entire susceptible target surface has proven to be important for preventing and treating infections. Moreover, in order to increase the user's acceptability, a drug delivered to the vagina must be retained in the vaginal canal at an effective concentration over an extended period of time. Achieving sustained local drug concentrations is a challenge because the vaginal epithelium is highly permeable to small molecules and also soluble drug dosage forms (gels, creams) can be excreted by intraperitoneal pressure and walking. Finally, the drug delivery method must be safe and non-toxic to the vaginal epithelium. The distribution, retention, and safety profile of the vaginal dosage form can result in a significant increase in efficacy and a decrease in side effects caused by systemic treatments that are primarily ineffective for cervical infections and diseases.

Nanoparticles have received considerable attention due to their ability to deliver all sustained topical drugs to the vagina. However, the mucus layer that coats the vaginal epithelium exists as a barrier to achieve uniform distribution and long-term retention in the vaginal canal. Mucus efficiently traps most particulates, including conventional polymer nanoparticles (CP), through both adhesion and steric interactions. The effectiveness of the mucus to entrap exogenous pathogens and particulates means that the CP will be immediately captured upon contact with the lumenal mucus layer, preventing entry into the mucosal folds, and thus protection. Particles and pathogens entrapped in the superficial luminal mucus layer are expected to be rapidly cleared from tissue, which limits the residence time of mucoadhesive substances such as CP.

By mimicking the evolved bias to penetrate the mucus barrier, mucus-penetrating particles (MPP) have recently been engineered for mucosal drug delivery by coating CPs with exceptionally high density, low molecular weight poly(ethylene glycol) (PEG). MPP diffuses through the human cervical mucus (CVM) at a rate comparable to its theoretical diffusion through water. Here, the MPP penetrates into the deepest mucus layer, including the mucus cleared more slowly in the mucosal folds, thereby releasing the drug in an optimal location for efficient tissue absorption, thereby providing enhanced distribution and increased retention in the vagina in vivo. It was intended to test the hypothesis that In addition to the conventional progestin-induced diestrus phase (DP) mouse model, the use of an estradiol-induced estrous phase (IE) mouse model is introduced, wherein mouse CVM (mCVM) is further subdivided into human CVM (hCVM). It closely mimics and thus provides a more human-like model for developing and transforming MPPs for human use.

Carboxylic acid-coated, fluorescent polystyrene nanoparticles (PS-COOH) were prepared as previously reported (YY Wang, SK Lai, JS Suk, A. Pace, R. Cone, J. Hanes, Angew Chem). Int Ed Engl 47, 9726-9729 (2008)]; [SK Lai, DE O'Hanlon, S. Harrold, ST Man, YY Wang, R. Cone, J. Hanes, Proc Natl Acad Sci USA 104, 1482-1487 (2007)]), prepared with MPP by covalently attaching a dense coating of low molecular weight PEG. Furthermore, biodegradable MPP (BD-MPP), as previously reported (M. Yang, SK Lai, YY Wang, W. Zhong, C. Happe, M. Zhang, J. Fu, J. Hanes , Biodegradable Nanoparticles Composed Entirely of Safe Materials that Rapidly Penetrate Human Mucus. Angew Chem Int Ed Engl 50, 2597-2600 (2011)]), poly(lactic-co-glycolic acid) (PLGA) core and physically adsorbed PEG coating It is formulated with water because the biodegradable particles can be loaded with drugs and are suitable for administration to humans. PS-COOH and PLGA nanoparticles have a highly negative surface charge, which is almost neutralized when densely coated with PEG. Nanoparticles were prepared as previously described (SK Lai, DE O'Hanlon, S. Harrold, ST Man, YY Wang, R. Cone, J. Hanes, Proc Natl Acad Sci USA 104, 1482-1487 (2007)). ]), which was determined to be well-coated by measuring the zeta potential (Table 12). A zeta potential more neutral than -10 mV was previously shown to be essential for mucus-penetrating properties among hCVMs (YY Wang, SK Lai, JS Suk, A. Pace, R. Cone, J. Hanes, Addressing the PEG). mucoadhesivity paradox to engineer nanoparticles that "slip" through the human mucus barrier. Angew Chem Int Ed Engl 47, 9726-9729 (2008)]). To ensure that MPP is mucus-penetrating in native estrous mCVM, particles were administered intravaginally to estrous mice. The entire vagina was then excised and opened to visualize the migration of hundreds of individual particles by multi-particle tracking (MPT) methods (J. Suh, M. Dawson, J. Hanes, Adv Drug Deliv Rev 57, 63-78). (2005)]). The particle trajectory for MPP was indicative of rapid diffusion through the watery pores in mCVM, while the migration of uncoated PS-COOH nanoparticles (CP) was smaller than the particle diameter (~100 nm) (Fig. 14A). . The ensemble averaged mean square displacement (<MSD>) of MPP in mCVM was reported for MPP in hCVM (SK Lai, YY Wang, K. Hida, R. Cone, J. Hanes, Nanoparticles reveal that human cervicovaginal mucus is riddled with pores larger than viruses. ) corresponds to an ensemble-averaged effective diffusivity (<Deff>) that is only ~8 times slower than

Based on the measured D _eff for individual particles, with Pick's second law of diffusion about half of the MPP will diffuse a 100 μm-thick layer of mCVM after about 4 h ( FIG. 14D ), while CP even after 24 h was evaluated to have no recognizable penetration. _{The D eff} values for CP on a time scale of 1 s corresponded to MSD values smaller than the particle diameter (dotted line, FIG. 14C ), indicating probably not particle diffusion but thermal fluctuations of particles adhering to mucin fibers. Overall, the transport behavior of both MPP and CP in estrous mCVM was very similar to its transport behavior in hCVM.

Synchronizing a number of mice in estrus with respect to the residence study required hormonal treatment. Particle transport behavior was tested in IE mice and treated with estradiol (which has been commonly used to induce estrus-like behavior in many animal models) (JR Ring, The estrogen-progesterone induction of sexual receptivity in the spayed female mouse. Endocrinology 34, 269-275 (1944)] and [CA Rubio, The exfoliating cervico-vaginal surface. II. Scanning electron microscopical studies during the estrous cycle in mice. Anat Rec 185, 359-372 (1976)]), It was confirmed that MPP and CP transport were not altered ( FIG. 15A ) prior to distribution and retention studies. Moreover, the BD-MPP transport behavior was indistinguishable from MPP in IE mucus ( FIG. 15B ).

We then investigated whether the ability to penetrate mucus rapidly in estrus mice and IE mice would result in a faster and more uniform vaginal distribution of MPP compared to CP. MPP and CP were applied to a hypotonic medium to mimic the way in which osmotically driven water flow (advective transport) rapidly transports nutrients from the small intestine lumen to the basal epithelial surface. Ten minutes after particle administration, the entire vagina was dissected and cell nuclei were stained. CP aggregated in the luminal mucus and did not penetrate the vaginal mucosal folds (Figure 16). In contrast, MPP formed a continuous particle layer that coated the entire vaginal epithelium, including all surfaces of the mucosal folds. MPPs penetrated the mucus over -100 μm through advection within 10 minutes compared to the ~4 hours they would take to diffuse its distance through the mucus ( FIG. 14D ). This behavior was also consistent with BD-CP and BD-MPP, and CP and MPP, administered to IE mice ( FIG. 16 ). Videos illustrating the movement of MPPs through hCVM through muco-adhesive CPs can be found in video 1 (no induction, diffusion) and video 2 (with flow, advection).

To quantify the difference in the distribution of MPP and CP, fluorescence images were obtained from freshly excised, open, and planarized mouse vaginal tissue. As shown in Figure 17, attachment of CPs to the luminal vaginal mucus layer created “stripes” of mucus, where particles alternated with dark “stripes” of mucus without particles, the latter being , corresponding to the mucosal folds that open when the vaginal tissue is flattened. In contrast, transport of MPP towards the epithelium and into the mucosal folds created a continuous particle coating on the planarized vaginal surface ( FIG. 17 ). Quantification of fluorescence on vaginal and cervical tissue showed that 88% of the smoothed vaginal surface and 87% of the cervix were densely coated with MPP, while only 30% of the vaginal surface and 36% of the cervical surface were coated with CP. indicated as Upon further examination at higher magnification of the darker areas of the vaginal and cervical surfaces, a continuous, less concentrated coating of MPP was revealed ( FIGS. 17-18 , insets), which resulted in near complete coverage of the vaginal and cervical epithelium. means there is For CP, no less-concentrated coating was found at higher magnifications ( FIGS. 17-18 , insets). A similar trend was found with BD-MPP with 85% vaginal coverage and 86% cervical coverage, as well as with BD-CP with 31% vaginal coverage and 27% cervical coverage ( FIGS. 17-18 ).

Next, it was attempted to determine whether improved distribution of BD-MPP could improve the delivery of small molecules compared to the gel dosage form. The lipophilic molecules are likely to enter the first surface they come in contact with and not contact the cells in the mucosal folds. Conversely, hydrophilic molecules can rapidly diffuse through the vaginal epithelium and be transported by blood and lymph circulation to be cleared, resulting in short-term coverage. BD-MPP was loaded with a fluorescent, water-soluble small molecule, fluorescein isothiocyanate (FITC) as a model drug (FITC/MPP). To mimic conventional vaginal delivery, soluble FITC (FITC/gel) was administered in universal vaginal placebo gel hydroxyethylcellulose (HEC). Twenty-four hours after administration to estrous mice, vaginal tissue was excised and flattened to expose the vaginal folds. The patch of FITC coated 42% of the vaginal surface upon administration as FITC/gel, while FITC/MPP provided a well-retained FITC coating of 87% of the vaginal surface even 24 h after particle administration (Figure 22). ).

To further characterize the effect of the mucus barrier, we conducted removal of vaginal mucus by lavage prior to particle administration (Y. Cu, CJ Booth, WM Saltzman, In vivo distribution of surface-modified PLGA nanoparticles following Journal of Controlled Release 156, 258-264 (2011)] and [KA Woodrow, Y. Cu, CJ Booth, JK Saucier-Sawyer, MJ Wood, WM Saltzman, Intravaginal gene silencing using biodegradable polymer nanoparticles densely loaded with small-interfering RNA. Nat Mater 8, 526-533 (2009)]) significantly improved CP distribution, indicating that its mucoadhesive properties prevented uniform distribution in the vagina ( FIG. 19 ).

It was then intended to determine the vaginal retention of MPP compared to mucoadhesive CP using our IE model. Fluorescent MPP and CP were administered intravaginally to IE mice. At the specified time points, the entire reproductive organs (vaginal and uterine horn) were excised and quantitatively analyzed by fluorescence imaging ( FIG. 21A ). After an initial decrease in particle fluorescence similar for MPP and CP (probably due to an initial “squeeze out” preceding mucus penetration), the remaining amount of MPP remained constant at approximately 60% ( FIG. 21B ). . In contrast, the amount of CP decreased steadily over time to 10% (6 h). Importantly, although CPs were distributed along the length of the vagina, this longitudinal coverage did not indicate that CPs penetrate the mucus to the epithelium, nor did they reach the surface inside the vaginal folds, as shown in FIG. 16 . does not

The immune system is sensitive to mucosal surfaces (OP Mestecky J, McGhee JR, and Lambrecht BN, Mucosal Immunology. (Elselvier Academic Press, Burlington, ed. Third, 2005)), particularly living cells, such as the columnar epithelium of the human cervical endometrium. It is highly active in those with a surface covered with The inflammatory action of nanoparticles was pre-treated with Depo-Provera, a long-acting progestin treatment that synchronizes mice in the estrus interphase, during which the vaginal epithelium becomes thin and covered with living cells. Mice were used to investigate. In contrast, in estrus, the mouse vagina thickens with 4 to 7 cell layers to about 12 cell layers and the epithelial surface is protected by many layers of apoptotic and apoptotic cells (Biology of the laboratory mouse. Edited by George D. Snell. Dover Publications, Inc., New York, 1956 (Reprint of first edition, 1941). [Journal of the American Pharmaceutical Association 45, 819-819 (1956)). Moreover, the vaginal epithelium of progestin-induced estrus interphase (DP) mice has an increased immune cell population, which leads to an enhanced acute inflammatory response; Estrus, on the other hand, is characterized by the absence of immune cells (C. H. Hubscher, D. L. Brooks, J. R. Johnson, A quantitative method for assessing stages of the rat estrous cycle. Biotech Histochem 80, 79-87 (2005)). Depo-Provera synchronizes mice to an estrus interphase-like state for days to weeks, which is important for experiments to last for more than 24 h.

The potential toxic effects of intravaginally administered nanoparticles were investigated using standard hematoxylin and eosin (H&E) staining. Nonoxynol-9 (N9), a nonionic cleanser known to cause vaginal toxicity (G. Ramjee, A. Kamali, S. McCormack, The last decade of microbicide clinical trials in Africa: from hypothesis to facts. AIDS 24 Suppl 4, S40-49 (2010)) was used as a positive control and PBS (saline) was used as a negative control. The same (BD-) MPP and (BD-) CP used in the distribution and retention studies were tested for toxicity. As expected, N9 induced acute inflammation at 24 h that did not appear after PBS treatment ( FIG. 23 ). CP, such as N9, induced pronounced neutrophil infiltration into the lumen, whereas MPP did not induce this inflammatory action ( FIG. 23 , arrowheads).

Recent studies have shown that in response to certain vaginal products, the vaginal epithelium can secrete immune mediators that can enhance susceptibility to sexually transmitted infections (JE Cummins, Jr., GF Doncel, Biomarkers of cervicovaginal inflammation). for the assessment of microbicide safety. Sex Transm Dis 36, S84-91 (2009)] and [SS Wilson, N. Cheshenko, E. Fakioglu, PM Mesquita, MJ Keller, BC Herold, Susceptibility to genital herpes as a biomarker predictive. of increased HIV risk: expansion of a murine model of microbicide safety. Antivir Ther 14, 1113-1124 (2009)]). Therefore, it is important that the vaginal product does not induce such an immune response, especially after repeated administration. Since our ultimate goal was to test MPP for protection against HSV-2, an MPP formulation containing acyclovir monophosphate (ACVp) was recently used in the tenofovir clinical trial, N9, HEC Placebo gel, PBS, and gel vehicle (TFV vehicle) were compared. Nanoparticles and control formulations were administered vaginally to Depo Provera-treated mice daily for 7 days. Vaginal lavages from each mouse were collected on day 8 and assessed for cytokines found to be elevated in response to epithelial stimulation: interleukin 1β (IL-1β), interleukin 1α (IL-1α), tumor necrosis factor a (TNF). -α), and interleukin 6 (IL-6). Both IL-1α and IL-1β levels were found to be enhanced in response to both TFV vehicle and N9 solution ( FIG. 24 ). This was not surprising, given that IL-1α and IL-1β are secreted by the vaginal epithelium in response to injury (JE Cummins, Jr., GF Doncel, Biomarkers of cervicovaginal inflammation for the assessment of microbicide safety. Sex Transm Dis 36, S84-91 (2009)]). In contrast, cytokine levels associated with ACVp-MPP were equivalent to those associated with HEC placebo gel ( FIG. 24 ) (which has been used in clinical trials without any associated increase in susceptibility to infection) (QA Karim, SSA Karim , JA Frohlich, AC Grobler, C. Baxter, LE Mansoor, ABM Kharsany, S. Sibeko, KP Mlisana, Z. Omar, TN Gengiah, S. Maarschalk, N. Arulappan, M. Mlotshwa, L. Morris, D. Taylor , CT Grp, Effectiveness and Safety of Tenofovir Gel, an Antiretroviral Microbicide, for the Prevention of HIV Infection in Women. Science 329, 1168-1174 (2010); and D. Tien, RL Schnaare, F. Kang, G. Cohl, TJ McCormick, TR Moench, G. Doncel, K. Watson, RW Buckheit, MG Lewis, J. Schwartz, K. Douville, JW Romano, in vitro and in vivo characterization of a potential universal Placebo designed for use in vaginal microbicide AIDS Res Hum Retroviruses 21, 845-853 (2005)]). There was no detectable elevation of either IL-6 or TNF-α associated with any vaginal treatment compared to untreated controls.

Finally, it was investigated whether an improved distribution, retention, and toxicity profile of MPP would result in improved protection against vaginal HSV-2 challenge in mice. Depot probera treatment significantly increased the vaginal susceptibility of mice to infection, and candidate microbicides, even when administered immediately prior to infection inoculum, provided only partial protection in the mouse model used here (SL Achilles). , PB Shete, KJ Whaley, TR Moench, RA Cone, Microbicide efficacy and toxicity tests in a mouse model for vaginal transmission of Chlamydia trachomatis. Sex Transm Dis 29, 655-664 (2002); and L. Zeitlin, KJ Whaley, TA Hegarty, TR Moench, RA Cone, Tests of vaginal microbicides in the mouse genital herpes model. Contraception 56, 329-335 (1997)]). Moreover, some vaginal excipients actually increase susceptibility to infection in this model (RA Cone, T. Hoen, X. Wong, R. Abusuwwa, DJ Anderson, TR Moench, Vaginal microbicides: detecting toxicities in vivo that BMC Infect Dis 6, 90 (2006)] and [TR Moench, RJ Mumper, TE Hoen, M. Sun, RA Cone, Microbicide excipients can greatly increase susceptibility to genital herpes transmission in the mouse. Dis 10, 331 (2010)]). Testing ACVp to block vaginal transmission of HSV-2 infection was chosen because acyclovir provides viral suppression in animals with multiple daily repeated doses (ER Kern, Acyclovir Treatment of Experimental [ER Kern, Acyclovir Treatment of Experimental] Genital Herpes-Simplex Virus-Infections. Am J Med 73, 100-108 (1982)]). However, as a result of a single vaginal pretreatment with 50 mg/mL (5%) ACVp in guinea pigs, 70% of animals compared to controls were infected (ER Kern, J. Palmer, G. Szczech, G. Painter). , KY Hostetler, Efficacy of topical acyclovir monophosphate, acyclovir, or penciclovir in orofacial HSV-1 infections of mice and genital HSV-2 infections of guinea pigs. Nucleos Nucleot Nucl 19, 501-513 (2000)]). ACVp thus provided a test case to determine whether MPP could significantly improve protection by water-soluble and rapidly metabolized drugs by prolonging treatment-related drug concentrations after a single application. Moreover, the mechanism of action of nucleotide analogues, such as ACVp, is the prevention of intracellular viral replication, and thus successful protection would imply efficient uptake and retention in susceptible target cell populations in the vaginal and cervical mucosa.

ACVp nanoparticles were formulated with the same mucosal-inert coating used for all other studies. The size and ζ-potential of ACVp-MPP was similar to that of polystyrene (PS)-based MPP (Table 12). Soluble ACVp or ACVp-MPP was administered to the vagina of mice 30 minutes prior to challenge with HSV-2. Soluble drug administered at the same concentration as ACVp-MPP (1 mg/mL) was ineffective in protecting mice from viral infection (84.0% infected compared to 88.0% of the control group), whereas only mice in the ACVp-MPP group were 46.7% were infected (Table 11). Groups of mice given soluble drug at 10 times the concentration of ACVp-MPP were still infected at a rate of 62.0% (drug in PBS) or 69.3% (drug in water). When the soluble drug was compared to ACVp-MPP in the same vehicle (pure water), the soluble drug was significantly less protective than ACVp-MPP even at 10-fold higher concentrations (Table 11). To study the distribution and retention of nanoparticles on the vaginal mucosal surface and the effect of repeated dosing, 6-8 week old CF-1 mice (Harlan) were used. Mice were housed in a reverse photoperiodic facility (12 h light/12 h dark). For spontaneous cycle estrus, mice were selected for external estrus appearance and confirmed at dissection (AK Champlin, DL Dorr, AH Gates, Determining the stage of the estrous cycle in the mouse by the appearance of the vagina. Biol Reprod 8, 491-494 (1973)]; [E. Allen, The oestrous cycle in the mouse. American Journal of Anatomy 30, 297-371 (1922)]). For hormone-induced estrus (IE), mice were acclimated for 3 weeks and injected subcutaneously with 100 μg of 17-β estradiol benzoate (Sigma) 2 days before the experiment. Numerous studies have demonstrated that treatment with estradiol induces a "estrus-like" condition with similar epithelial features and vaginal cell populations (CG Rosa, JT Velardo, Histochemical localization of vaginal oxidative enzymes and mucins in rats treated). Ann NY Acad Sci 83, 122-144 (1959)]; [CA Rubio, The exfoliating cervico-vaginal surface. II. Scanning electron microscopical studies during the estrous cycle in mice. Anat Rec 185, 359-372 (1976)]; [AE Gillgrass, SA Fernandez, KL Rosenthal, C. Kaushic, estradiol regulates susceptibility following primary exposure to genital herpes simplex virus type 2, while progesterone induces inflammation. Journal of Virology 79, 3107-3116 (2005)] ). For vaginal toxicity and cytokine release, mice were injected subcutaneously with 2.5 mg of Depo-Provera (Medroxyprogesterone Acetate, 150 mg/mL) (Pharmacia & Upjohn Company) 7 days prior to the experiment.

Water was used as the hypotonic medium for all particle solutions. For ex vivo tracking, 5 μL of particles were administered intravaginally. After approximately 10 minutes, the vagina was removed and carefully sliced open and laid flat. A cover slip was placed on top and the entire tissue was placed in a custom-made well so that the tissue was in contact with the mucus without deformation. The wells were approximately 1 mm x 0.5 mm rectangles cut from three layers of electrical tape attached to a standard glass slide. The coverslips were sealed with super adhesive around the edges and immediately imaged to prevent drying.

Mice were anesthetized prior to experimental procedures, including sacrifice by cervical dislocation. For all experiments, mice were prevented from self-grooming by a collar of weak adhesive tape around the abdomen and inter-grooming by housed in individual cages. did.

For conventional mucoadhesive particles (CP), fluorescent, carboxyl (COOH)-modified polystyrene (PS) nanoparticles (Molecular Probes) with a size of 100 nm were used. These particles are characterized by a negatively charged surface at neutral pH (Table 12). To prepare mucus-penetrating particles (MPP), 5-kDa amine-modified PEG (Creative PEGworks) via standard 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide coupling reaction ) covalently modified CP. Particle size and ξ-potential were determined by dynamic light scattering and laser Doppler anemometers, respectively, using a Zetasizer Nano ZS90 (Malvern Instruments). The size measurements were 25 at a scattering angle of 90°. C. The sample was dissolved in 10 mM NaCl solution (pH 7) and the measurement was carried out according to the instrument instructions.The PEG conjugation was confirmed using the near-neutral ξ-potential measured by laser Doppler anemometer. did.

For biodegradable particles, PLGA acid 2A (50:50 Lakeshore Biomaterials), Lutrol F127 (BASF), and poly(vinyl alcohol) (PVA 25 kDa, Polysciences) ) was used. Alexa Fluor 555 was chemically conjugated to PLGA and nanoparticles were prepared by nanoprecipitation as previously described (M. Yang, SK Lai, YY Wang, W. Zhong, C. Happe, M. Zhang). , J. Fu, J. Hanes, Biodegradable Nanoparticles Composed Entirely of Safe Materials that Rapidly Penetrate Human Mucus. Angew Chem Int Ed Engl 50, 2597-2600 (2011)]). Briefly, 10 mg/mL of labeled PLGA was dissolved in acetone or THF (with or without 2 mg FITC) and added dropwise to 40 mL of aqueous surfactant solution. After stirring for 2 h, the particles were filtered through a 5-μm syringe filter, collected by centrifugation (Sorvall RC-6+, ThermoScientific) and washed. Particle size and ξ-potential were determined as described.

ACVp-MPP was prepared by dissolving ACVp in ultrapure water containing Lutrol F127. Zinc acetate was added in a molar ratio of 5:1 ACVp:Zn to chelate the ACVp and render it water-insoluble, then immediately flash frozen and freeze-dried. Particle characterization was performed after restoration. Using ultrapure water prior to administration, the powder was reconstituted to a final concentration of 1 mg/mL ACVp and 0.8 mg/mL Lutrol. Soluble ACVp was titrated with NaOH if necessary to reach pH 6-7. Particle size and ξ-potential were determined as described.

Detection of fluorescent particles in ex vivo vaginal tissue samples using a silicon-enhanced targeting camera (VE-1000, Dage-MTI) equipped with an inverted epifluorescence microscope equipped with a 100X oil immersion objective (numerical aperture 1.3). The trajectory was recorded. Movies were captured using Metamorph software (Universal Imaging Corp.) at a temporal resolution of 66.7 ms for 20 s. The trajectories of n > 130 particles were analyzed for each experiment and from different mice. Three independent experiments were performed using the tissue: The coordination of the particle center of gravity was transformed into a time-averaged mean square displacement (<MSD>), which was

<Δr ² (τ)> = [x(t+τ) - x(t)] ² + [y(t+τ) - y(t)] ²

(where τ is the time scale (or time lag), x and y are the corresponding particle configurations at time t, and Δr ² is the MSD). Particle MSD and effective diffusivity (D _eff ) were calculated using this equation as previously demonstrated (SK Lai, DE O'Hanlon, S. Harrold, ST Man, YY Wang, R. Cone, J. Hanes, Rapid transport of large polymeric nanoparticles in fresh undiluted human mucus.Proc Natl Acad Sci USA 104, 1482-1487 (2007)];BC Tang, M. Dawson, SK Lai, YY Wang, JS Suk, M. Yang, P. Zeitlin, MP Boyle, J. Fu, J. Hanes, Biodegradable polymer nanoparticles that rapidly penetrate the human mucus barrier. Proc Natl Acad Sci USA 106, 19268-19273 (2009)]). Calculated D _eff values were used for modeling of particle penetration through mucus slabs, as previously described (BC Tang, M. Dawson, SK Lai, YY Wang, JS Suk, M. Yang, P. Zeitlin, MP Boyle, J. Fu, J. Hanes, Biodegradable polymer nanoparticles that rapidly penetrate the human mucus barrier. Proc Natl Acad Sci USA 106, 19268-19273 (2009)]).

For distribution by mucus removal, prior to particle administration, mice were given a 2x vaginal wash with 50 μL of PBS and then swabbed once with a cotton-tipped applicator. Subsequently, 5 μL of either CP or MPP was administered intravaginally. The entire vagina is then removed and Tissue-Tek O.C.T. Compounds were frozen in (Sakura Finetek U.S.A., Inc.). Transverse sections were obtained at various points along the length of the tissue (between the vaginal opening and the cervix) using a Microm HM 500 M Cryostat (Microm International). . The thickness of the section was set to 6 μm to achieve a single cell layer thickness. The sections were then stained with ProLong Gold (Invitrogen) antifade reagent and DAPI to visualize cell nuclei and maintain particle fluorescence. Fluorescence images of the sections were obtained with an inverted fluorescence microscope. To quantify the nanoparticle distribution, 5 μL of either CP or MPP was administered intravaginally. Within 10 minutes, the vaginal tissue, including "blank" tissue, which had not been dosed with any particles, was opened by longitudinal slices and clamped between two glass slides and sealed tightly with super adhesive. This procedure completely flattened the tissue and exposed the wrinkles. "Blank" tissue was used to evaluate background tissue fluorescence levels to ensure that all images taken were well above the background level. Six fluorescence images at low magnification and one or more images at high magnification were taken from each tissue. The images were thresholded to draw a border around the fluorescence signal, and then the covered area was quantified using ImageJ software. Average coverage was determined for each mouse and then these values were averaged over groups of n≧3 mice. The cervix from each mouse was excised from the uterine horn and mounted using the same custom-made wells used for in vitro particle tracking. Wells were sealed with coverslips and background fluorescence levels were determined using blank tissue. One fluorescence image comprising almost the entire cervical surface was taken at low magnification above the tissue background level. These images were thresholded in the same way to determine the area covered by the particles. One or more higher magnification images were taken of each tissue to reveal individual particles.

Advection of MPP and CP was visualized using a custom capillary tube setup. A flat capillary tube (0.4 mm × 4 mm × 50 mm; VitroCom) was attached to a 1 mL tuberculin syringe (Becton Dickinson) through a single strand of flexible plastic tubing. The tubing was clamped at one end of the capillary and sealed using silicone grease. Fresh, dilution mixed with saline, followed by 3% (v/v) of ~500 nm uncoated (red fluorescence) and PEG-coated (green fluorescence) polystyrene beads (Invitrogen) in syringe and tubing Non-human CVM was loaded. PEG-coated beads were prepared as described above for the 100 nm MPP used in the mouse study. Approximately 80 µL of mucus was required to fill the capillary, taking care not to introduce air bubbles. Time lapse videos showing migration of MPPs and CPs within capillaries were recorded on a Zeiss LSM 510 confocal microscope (Carl Zeiss MicroImaging, LLC) with and without applied pressure. Recordings were made using a 40× objective.

FITC dye (Sigma-Aldrich) was mixed at 1 mg/mL in HEC gel favorably provided by T. Moench (Reprotect). Biodegradable MPP was prepared as described, loaded with FITC dye and suspended in 1% Lutrol F127. To assess distribution, either 10 μL of gel or particle solution was administered intravaginally. After 24 h, the vaginal tissue was removed, cut open and laid flat. The tissue was then mounted between two microscope slides and squeezed to smooth the mucosal folds. Background autofluorescence was determined from vaginal tissue by including "blank" tissue to ensure that the exposure setting used was necessarily indicative of the presence of FITC. Fluorescence images of dye distribution on planarized tissue surfaces were obtained using a Nikon E600 inverted microscope equipped with a 2× objective. These images were thresholded in the same way using ImageJ to determine the coverage area.

To assess nanoparticle retention, 5 μL of red fluorescent CP or MPP was administered intravaginally. Whole cervical canals were obtained at 0, 2, 4, and 6 h and placed in standard tissue culture dishes. For each condition and time point, n > 7 mice were used. Fluorescence images of tissues were obtained using a Xenogen IVIS spectral imaging device (Caliper Life Sciences). Quantification of fluorescence counts per unit area was calculated using Xenogen Living Image 2.5 software.

5 μL of particles or control solution was administered intravaginally to the DP mouse model. After 24 h, the entire cervical canal was obtained and fixed in 4% paraformaldehyde solution for 24 h. Tissues were placed in 70% ethanol and taken to The Johns Hopkins Reference Histology Laboratory for paraffin embedding and standard H&E staining.

20 μL of each test agent was administered intravaginally to the DP mouse model once a day for seven (7) days. HEC gel and N9 were provided by T. Moenchi (Reprotect), and TFV vehicle gel was provided favorably by C. Dezzutti (University of Pittsburgh). On day 8, each mouse was washed twice with 50 μL of PBS. Each wash sample was diluted with an additional 200 μL of PBS and centrifuged to remove the mucus plug. Remove the supernatant (200 μL) and remove 4 (IL-1β, IL-1α, TNF-α, and IL-6) Quantikine ELISA kits (R&D Systems, Inc. 50 for each The ELISA was performed according to the manufacturer's instructions.

All data are presented as means using standard error of the mean (SEM) specified. Statistical significance was determined by a two-tailed, Student's t test (α = 0.05) estimating unequal variance. For the HSV-2 challenge, statistical significance was determined using Fisher's exact test, a two-way distribution.

The female reproductive system is susceptible to a wide range of sexually transmitted infections (R. Mallipeddi, L. C. Rohan, Nanoparticle-based vaginal drug delivery systems for HIV prevention. Expert Opin Drug Deliv 7, 37-48 (2010)). Biological vulnerability, lack of female-controlled prophylaxis, and failure to negotiate condom use all contribute to male-to-female transmission worldwide (R. Mallipeddi, LC Rohan, Nanoparticle-based vaginal drug delivery systems for HIV prevention. Expert Opin Drug Deliv 7, 37-48 (2010)]; VM Ndesendo, V. Pillay, YE Choonara, E. Buchmann, DN Bayever, LC Meyer, A review of current intravaginal drug delivery approaches employed for the propylaxis of HIV/AIDS and prevention of sexually transmitted infections. AAPS PharmSciTech 9, 505-520 (2008)]). An easily administered, prudent, and effective method for protecting women against vaginal HIV, HSV-2, and other viral transmission could prevent millions of infections worldwide. After 11 unsuccessful biocide trials, CAPRISA 004 was the first to demonstrate partial protection against HIV with a microbicide (tenofovir) administered vaginally in a gel formulation (QA Karim, SSA Karim). , JA Frohlich, AC Grobler, C. Baxter, LE Mansoor, ABM Kharsany, S. Sibeko, KP Mlisana, Z. Omar, TN Gengiah, S. Maarschalk, N. Arulappan, M. Mlotshwa, L. Morris, D. Taylor , CT Grp, Effectiveness and Safety of Tenofovir Gel, an Antiretroviral Microbicide, for the Prevention of HIV Infection in Women. Science 329, 1168-1174 (2010)]). An important difference between previous generations of microbicides such as N9 and current generations of microbicides is the site of action. Many current-generation microbicides, such as the nucleotide analogs tenofovir and acyclovir monophosphate, act intracellularly to inhibit viral replication, while previous generations directly inactivated pathogens in the vaginal lumen. However, some previous-generation microbicides caused toxicity to the vaginal epithelium, which increased susceptibility to infection (OJ D'Cruz, FM Uckun, Dawn of non-nucleoside inhibitor-based anti-HIV microbicides. J Antimicrob Chemother 57 , 411-423 (2006)]).

To be maximally effective in vaginal drug delivery, topically delivered drugs must be uniformly distributed, maintained at sufficiently high concentrations, and remain in close proximity to the folds of the vaginal epithelium (mucosal folds) and cervical mucosa. Several techniques are used to observe the distribution of the gel and drug following vaginal administration, such as MRI (CK Mauck, D. Katz, EP Sandefer, MD Nasution, M. Henderson, GA Digenis, I. Su, R. Page, K. Barnhart, Vaginal distribution of Replens and KY Jelly using three imaging techniques. Contraception 77, 195-204 (2008)), gamma-scintigraphy (CK Mauck, D. Katz, EP Sandefer, MD Nasution, M. Henderson, GA Digenis, I. Su, R. Page, K. Barnhart, Vaginal distribution of Replens and KY Jelly using three imaging techniques. Contraception 77, 195-204 (2008); and BE Chatterton, S Penglis, JC Kovacs, B. Presnell, B. Hunt, Retention and distribution of two 99mTc-DTPA labeled vaginal dosage forms. Int J Pharm 271, 137-143 (2004)), colposcopy (N. Poelvoorde, H. Verstraelen, R. Verhelst, B. Saerens, E. De Backer, GL dos Santos Santiago, C. Vervaet, M. Vaneechoutte, F. De Boeck, L. Van Bortel, M. Temmerman, JP Remon, In vivo evaluation of the vaginal distribution and retention of a multi-particulate pellet fo rmulation. Eur J Pharm Biopharm 73, 280-284 (2009)), and fiber optics (CK Mauck, D. Katz, EP Sandefer, MD Nasution, M. Henderson, GA Digenis, I. Su, R. Page, K (Barnhart, Vaginal distribution of Replens and KY Jelly using three imaging techniques. Contraception 77, 195-204 (2008)]) have been used. Although these techniques are suitable for observing the gross distribution along the vaginal canal, they do not reveal the entry into the vaginal folds. Our study demonstrates that, although topical treatment can be well-distributed longitudinally along the vaginal canal, a large portion of the folded epithelium may remain untreated and unprotected. Such untreated surfaces may be responsible for the recent failure of several candidate biocides against HIV in clinical trials (CW Hendrix, YJ Cao, EJ Fuchs, Topical microbicides to prevent HIV: clinical drug development challenges. Annu Rev. Pharmacol Toxicol 49, 349-375 (2009)]). Moreover, when a fluid or gel is administered to the vagina, it is in direct contact with the rapidly shed outer luminal mucus layer. Mucoadhesive particles, such as CP, are entrapped in this superficial mucus layer and are thereby excluded from mucosal folds. In contrast, we have demonstrated that MPP is deeply penetrable into the mouse mucosal folds and, when delivered hypotically, provides complete coverage of the epithelium in only 10 minutes.

The diffusion of the particles is not fast enough to result in such a uniform epithelial coating within minutes. Diffusion over ~100 μm can take about several hours. However, the vaginal epithelium has a great ability to absorb fluids induced by the osmotic gradient. Absorption of water through the mucus barrier helps the MPP rapidly reach the entire epithelial surface by advection, where the drug payload can be released for optimal tissue absorption. In contrast, water absorption is not beneficial for CPs as they are adherently entrapped and immobilized in luminal mucus (Video 2).

Insufficient retention of therapeutically active compounds in the vaginal canal is another limiting factor for vaginal protection. For example, many vaginal spermicides provide protection for only 1 h (LJD Zaneveld, DP Waller, N. Ahmad, J. Quigg, J. Kaminski, A. Nikurs, C. De Jonge, Properties of a new , long-lasting vaginal delivery system (LASRS) for contraceptive and antimicrobial agents. J Androl 22, 481-490 (2001)]). Other vaginal products are not well-retained even after 6 h (RF Omar, S. Trottier, G. Brousseau, A. Lamarre, G. Alexandre, MG Bergeron, Distribution of a vaginal gel (Invisible Condom) before, during and after simulated sexual intercourse and its persistence when delivered by two different vaginal applicators: a magnetic resonance imaging study. Contraception 77, 447-455 (2008)]; [N. Poelvoorde, H. Verstraelen, R. Verhelst, B. Saerens, E. De Backer, GL dos Santos Santiago, C. Vervaet, M. Vaneechoutte, F. De Boeck, L. Van Bortel, M. Temmerman, JP Remon, In vivo evaluation of the vaginal distribution and retention of a multi-particulate pellet Eur J Pharm Biopharm 73, 280-284 (2009)]; BE Chatterton, S. Penglis, JC Kovacs, B. Presnell, B. Hunt, Retention and distribution of two 99mTc-DTPA labeled vaginal dosage forms. Int J Pharm 271, 137-143 (2004)]), requiring repeated dosing for adequate protection. Similarly, more than 90% of CPs were shedding from the vagina within 6 h because they did not penetrate deeply into the mucus layer. In contrast, provided MPP improved delivery of encapsulated model drug (FITC) for at least 24 h compared to soluble drug in gel formulation. Thus, MPP may provide a means to achieve potent once-daily topical vaginal administration for treatments such as microbicides for sexually transmitted diseases.

In previous attempts to develop mucosal drug delivery systems for the vaginal canal, various “preparative treatments” have been used to reduce the mucus barrier. Prior to administration of the mucoadhesive delivery vehicle, fluid (Y. Cu, CJ Booth, WM Saltzman, In vivo distribution of surface-modified PLGA nanoparticles following intravaginal delivery. J Control Release, (10.1016/j.jconrel.2011.06. 036)]; )]; [T. Kanazawa, Y. Takashima, S. Hirayama, H. Okada, Effects of menstrual cycle on gene transfection through mouse vagina for DNA vaccine. Int J Pharm 360, 164-170 (2008)]; Kanazawa, Y. Takashima, Y. Shibata, M. Tsuchiya, T. Tamura, H. Okada, Effective vaginal DNA delivery with high transfection efficiency is a good system for induction of higher local vaginal immune responses. 1463 (2009)]), Swab (KA Woodrow, Y. Cu, CJ Booth, JK Saucier-Sawyer, MJ Wood, WM Saltzman, Intravaginal gene silencing using biodegradable polymer nanoparticles densely l loaded with small-interfering RNA. Nat Mater 8, 526-533 (2009)]; [AS Kask, X. Chen, JO Marshak, L. Dong, M. Saracino, D. Chen, C. Jarrahian, MA Kendall, DM Koelle, DNA vaccine delivery by densely-packed and short microprojection arrays to skin protects against vaginal HSV -2 challenge. Vaccine 28, 7483-7491 (2010)]), or degrading enzymes (MM Seavey, TR Mosmann, Estradiol-induced vaginal mucus inhibits antigen penetration and CD8(+) T cell priming in response to intravaginal immunization. Vaccine 27, 2342-2349 (2009)]) appears to have been essential for the drug or gene delivery achieved in these studies. Here, it was found that swab pretreatment in addition to washing significantly improved the distribution of CP in the vagina, which could cause the particles to coat the epithelium similarly to MPP ( FIG. 19 ). Barrier-removing pretreatment is impractical for use in humans and may be inappropriate, particularly for microbicides intended to prevent sexually transmitted diseases. Healthy CVM itself is a somewhat effective barrier to viral infection (SK Lai, K. Hida, S. Shukair, YY Wang, A. Figueiredo, R. Cone, TJ Hope, J. Hanes, Human immunodeficiency virus type 1 is trapped by acidic but not by neutralized human cervicovaginal mucus. J Virol 83 , 11196-11200 (2009)]). It has been shown that effective epithelial coverage can be achieved by using MPP without the need to degrade or remove the mucus barrier.

PEG coatings have been widely used to develop polymeric drug carriers that are not readily recognized by the immune system (BC Tang, M. Dawson, SK Lai, YY Wang, JS Suk, M. Yang, P. Zeitlin, MP Boyle, J. Fu, J. Hanes, Biogradable polymer nanoparticles that rapidly penetrate the human mucus barrier. Proc Natl Acad Sci USA 106, 19268-19273 (2009)]). It has been demonstrated that a dense PEG coating results in MPPs rapidly penetrating mucus without inducing inflammation in the mouse vaginal canal. In contrast, administration of uncoated CP resulted in an acute inflammatory response similar to administration of N9. Moreover, the cytokine levels associated with daily administration of MPP were indistinguishable from the HEC placebo gel. Elevated levels of IL-1α and IL-1β, associated with epithelial damage, occurred after daily administration of both N9 and TFV vehicle gels. The tenofovir-containing form of this gel has been shown to have complete protection against HIV in a tissue explant model, and complete protection that occurs despite visible epithelial shedding (LC Rohan, BJ Moncla, RP Kunjara Na Ayudhya, M. Cost, Y. Huang, F. Gai, N. Billitto, JD Lynam, K. Pryke, P. Graebing, N. Hopkins, JF Rooney, D. Friend, CS Dezzutti, In vitro and ex vivo testing of tenofovir shows it is effective as an HIV-1 microbicide. PLoS One 5, e9310 (2010)]). Previous studies suggest that glycerol in TFV gels may be responsible for the toxicity observed in mice (TR Moench, RJ Mumper, TE Hoen, M. Sun, RA Cone, Microbicide excipients can greatly increase susceptibility to genital herpes) transmission in the mouse. BMC Infect Dis 10, 331 (2010)]).

Mice are useful animal models for developing vaginal products, but there are key differences in vaginal physiology between mice and humans. First, in contrast to the 28-day human menstrual cycle, the estrous cycle occurs over a period of 4 to 5 days. Throughout the four phases of the mouse estrus cycle, there is significant growth followed by disruption of the epithelium, while there is relatively little change in the human vaginal epithelium over the menstrual cycle (BG Smith, EK Brunner, The structure of the human vaginal mucosa). in relation to the menstrual cycle and to pregnancy. American Journal of Anatomy 54, 27-85 (1934)]; epithelium Obstetrics & Gynecology 102, 571-582 (2003)]). The late pre- and early estrous phases of the mouse estrus cycle are most similar to those of the human vaginal epithelium (BG Smith, EK Brunner, The structure of the human vaginal mucosa in relation to the menstrual cycle and to pregnancy. American Journal of Anatomy 54 , 27-85 (1934)]; [AW Asscher, CH De Boer, CJ Turner, Cornification of the human vaginal epithelium. J Anat 90, 547-552 (1956)). At these stages, there is significant bacterial colonization, including peaks in the presence of Lactobacillus (HM Cowley, GS Heiss, Changes in Vaginal Bacterial Flora During the Oestrous Cycle of the Mouse. Microbial Ecology in Health and Disease 4, 229-235 (1991)]). In addition, estradiol effects cause active secretion of mucus (HM Cowley, GS Heiss, Changes in Vaginal Bacterial Flora During the Oestrous Cycle of the Mouse. Microbial Ecology in Health and Disease 4, 229-235 (1991)) [LB Corbeil, A. Chatterjee, L. Foresman, JA Westfall, Ultrastructure of cyclic changes in the murine uterus, cervix, and vagina. Tissue Cell 17, 53-68 (1985)]; [CG Rosa, JT Velardo, Histochemical] localization of vaginal oxidative enzymes and mucins in rats treated with estradiol and progesterone. turned out to be Therefore, the IE mouse model is considered to be a valuable model in addition to the DP model commonly used to investigate vaginal delivery methods. Although estradiol can be used to synchronize mice in estrus, it does not "stop" them in estrus. They continue the cycle, while DP treatment can arrest mice in an estrus interphase-like phase for days to weeks (C. Kaushic, AA Ashkar, LA Reid, KL Rosenthal, Progesterone increases susceptibility and decreases immune responses to genital herpes infection. J Virol 77, 4558-4565 (2003)]).

It has been shown that MPP can rapidly penetrate human cervix and mouse vaginal mucus and that MPP significantly improves the rate and uniformity of coverage and residence time compared to conventional mucoadhesive nanoparticles. CP elicited an acute inflammatory response similar to the known irritant N9, but similar to a placebo gel, and MPP did not elicit any detected inflammatory response. MPP administered vaginally loaded with acyclovir monophosphate was more effective in protecting mice against vaginal HSV-2 infection than the soluble drug, even at 10-fold higher soluble drug concentrations. These results motivate further development of MPPs for the prevention and treatment of sexually transmitted infections, contraception, and treatment of other cervical disorders, for safe and effective vaginal drug delivery.

Example 8

This non-limiting example shows the relationship between the relative velocity of polystyrene (PS) particles coated with ^{Pluronic ® F127 in mucus and the density of Pluronic ® F127 on the particle surface.}

In one set of experiments, an aqueous dispersion (200 nm, 0.5% w/v) of carboxylated PS nanoparticles was equilibrated in the presence of ^{various concentrations of Pluronic ® F127 at room temperature for at least 24 hours. The density of Pluronic ®} F127 on the surface of the obtained PS / Pluronic ^® F127 nanoparticles was quantified as follows. The PS/Pluronic ^® F127 mixture was ultracentrifuged to complete sedimentation of the particles. As a result, Pluronic ^® F127 bound to PS precipitated together with the particles; ^{Pluronic ®} F127 not bound to PS remained in the supernatant. The concentration ^{of Pluronic ®} F127 (C _{F127, free} ) in the supernatant obtained was determined by gel permeation chromatography (GPC). In this experiment, an Agilent 1100 HPLC system equipped with a G1362A refractive index detector and an analytical Agilent PLgel 5 μm Mixed-C column were used. The concentration of bound Pluronic ^® F127 ( C _{F127, binding} ) was calculated:

C _{F127, bond} = C _F127 -C _{F127, free}

where C _F127 is the total concentration of ^{Pluronic ®} F127 present in the mixture. ^{Then, the number of Pluronic ®} F127 molecules per PS surface area (F127/nm 2 ) was calculated as follows:

where N _A is Avogadro's number, C F _{127, binding} is the molar concentration (in moles/L) of bound Pluronic ^® F127, and SA is the ratio of PS particles calculated from the manufacturer's (Invitrogen) specifications is the surface area (in nm2/g) and C _PS is the mass concentration (in g/L) of PS in the mixture. The number average molecular weight of ^{Pluronic ®} F127 specified by the manufacturer (BASF) was used in the calculations.

The ability of PS/Pluronic ^® F127 particles to penetrate mucus was measured as a relative velocity in the mucus of the human cervix using fluorescence microscopy and multi-particle tracking software as described in Examples 1 and 4-6. In particular, samples were particles of interest, negative controls were 200 nm fluorescent carboxylate-modified polystyrene particles without polymer coating, and positive controls were 200 nm fluorescent polystyrene particles with a dense coating of 2 or 5 kDa PEG (which were fixed with reduced mucoadhesive behavior). Samples, negative controls, and positive controls were differentiated from each other by their fluorescence color.

Fluorescence microscopy and multi-particle tracking software were used to characterize the relative velocity of particles in the mucus of the human cervix as described in Examples 1 and 4-6. In a typical experiment, ≤0.5 μL of particle suspension was added to 20 μL of fresh cervical mucus along with positive and negative controls. Using a fluorescence microscope equipped with a CCD camera, a time of 66.7 milliseconds (15 frames/s) at 100× magnification from several regions within each sample for each type of particle, i.e. sample, negative control, and positive control. A 15 second(s) movie was captured at resolution. Then, using advanced image processing software, the individual trajectories of multiple particles were measured over a time scale of 3.335 s (50 frames) or longer.

The results shown in FIG. 25 indicated that the relative velocity of PS particles coated with ^{Pluronic ®} F127 in the mucus increased when the density ^{of Pluronic ® F127 molecules on the particle surface was increased.}

other embodiments

While several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily envision various other means and/or structures for carrying out the function and/or obtaining the result and/or one or more of the advantages described herein. , such changes and/or modifications are each considered to be within the scope of the present invention. More generally, one of ordinary skill in the art would mean that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and the actual parameters, dimensions, materials, and/or configurations will depend on the specific application or applications in which the teachings of the present invention are used. It will be readily understood that it will be determined by Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Accordingly, it is to be understood that the foregoing embodiments are presented by way of example only, and that the present invention may be practiced otherwise than as specifically described and claimed within the scope of the appended claims and their equivalents thereto. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. Moreover, any combination of two or more of such features, systems, articles, materials, kits, and/or methods is provided in the present invention unless such features, systems, articles, materials, kits, and/or methods are inconsistent with each other. are included within the scope of the invention.

As used herein in the specification and claims, the indefinite articles “a” and “an” should be understood to mean “at least one” unless expressly stated otherwise.

As used herein in the specification and claims, the phrase “and/or” means “one or both” of the elements so joined, i.e., present in combination in some cases and separately in other cases. elements are to be understood. Other elements may optionally be present other than those specifically identified by the "and/or" clause, whether or not related to those elements specifically identified, unless expressly stated otherwise. Thus, by way of non-limiting example, reference to "A and/or B" when used in conjunction with an open-ended language such as "comprising", in one embodiment, includes, in one embodiment, A without B (optionally B In addition to including elements); In another embodiment, B (optionally including elements other than A) without A; In another embodiment, it may refer to both A and B (optionally including other elements), and the like.

As used herein in the specification and claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, "or" or "and/or" is inclusive, i.e., includes at least one of the list or the number of elements, but also more than one, and optionally, additional It should be construed as including items not listed. Only terms explicitly stated otherwise, such as "only one of" or "exactly one of," or, as used in the claims, "consisting of," shall refer to including the number or list of exactly one element. will be. In general, the term "or," as used herein, is an exclusive alternative when preceded by a term of exclusiveness, such as "either", "one of", "only one of", or "exactly one of". (ie, "one or the other but not both"). "Consisting essentially of", when used in the claims, should have its ordinary meaning as used in the field of patent law.

As used herein in the specification and claims, the phrase “at least one”, with reference to a list of one or more elements, means at least one element selected from any one or more of the elements in the list of elements, but includes an element It is to be understood that the inclusion of at least one of each and every element specifically recited in the list of elements does not necessarily include, and does not exclude, any combination of elements in the list of elements. This definition also allows for elements other than the specifically identified element to optionally be present in the list of elements to which the phrase "at least one" refers, whether or not related to that specifically identified element. Thus, by way of non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently, “at least one of, A and/or B”) is an embodiment at least one A (and optionally including elements other than B) in which no B is present, optionally including more than one; in another embodiment, at least one B (and optionally including elements other than A), optionally including more than one, in which no A is present; in another embodiment, at least one A, optionally including more than one, and optionally, at least one B (and optionally including other elements), and the like, optionally including more than one.

In the claims, as well as in the specification, all transitional phrases such as "comprising", "including", "having", "having", "containing", "involving" ", "having," and the like are to be understood as open-ended, ie, meaning including, but not limited to. "Consisting solely of" and "consisting essentially of" must be the closed or semi-closed transitional phrases specified in section 2111.03 of the United States Patent and Trademark Office's Manual of Patent Examination Procedures, respectively.

Claims (33)

A composition comprising a plurality of coated nanoparticles,
Each coated nanoparticle
a core particle comprising a solid drug or a salt thereof; and
a coating comprising a surface-altering agent surrounding the core particle;
wherein the drug or salt thereof has a solubility in water of 1 mg/mL or less at 25° C. at any point over the pH range, and the drug or salt thereof constitutes at least 80 wt % of the core particles,
The surface-altering agent comprises a triblock copolymer comprising a hydrophilic block-hydrophobic block-hydrophilic block configuration, wherein the hydrophobic block comprises poly(propylene oxide) and has a molecular weight of at least 2 kDa, and wherein the hydrophilic block comprises poly( ethylene oxide) or poly(ethylene glycol), constituting at least 15 wt% of the triblock copolymer, the hydrophobic block is associated with the surface of the core particle, and the hydrophilic block is present on the surface of the coated nanoparticles to be coated renders the nanoparticles hydrophilic, and the surface-altering agent is present on the surface of the core particle at a density of at least ^{0.001 molecules/nm 2 ;}
the coated nanoparticles have an average size of greater than or equal to 100 nm and less than or equal to 1 μm;
The coated nanoparticles are mucus-penetrating,
composition. The composition of claim 1 , wherein the surface-altering agent is non-covalently adsorbed to the core particle. The composition of claim 1 , wherein the surface-altering agent is present on the surface of the coated nanoparticles at a density of at least ^{0.01 molecules/nm 2 .} The composition of claim 1 , wherein the hydrophilic blocks of the triblock copolymer constitute at least 30 wt % of the triblock copolymer. 5. The composition of claim 4, wherein the hydrophobic block portion of the triblock copolymer has a molecular weight of at least 3 kDa. 6. The triblock copolymer according to claim 5, wherein the triblock copolymer is poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) or poly(ethylene glycol)-poly(propylene oxide)-poly(ethylene glycol) phosphorus composition. The composition of claim 1 , wherein the poly(propylene oxide) block has a molecular weight of at least 3 kDa. The composition of claim 1 , wherein the surface-altering agent is present in solution at a concentration of at least 0.1% (w/v). The composition of claim 1 , wherein each of the core particles comprises a crystalline agent or a salt thereof. The composition of claim 1 , wherein each of the core particles comprises an amorphous agent or a salt thereof. The composition of claim 1 , wherein each of the core particles comprises a salt of a solid medicament. The composition according to claim 1, wherein the drug or a salt thereof is at least one of a therapeutic agent and a diagnostic agent. The composition according to claim 1, wherein the drug or a salt thereof is at least one of a small molecule, a peptide, a peptidomimetic, a protein, a nucleic acid, or a lipid. The composition according to claim 1, wherein the drug or a salt thereof has a water solubility of 0.1 mg/mL or less at 25°C. The composition of claim 1, wherein the drug or salt thereof constitutes at least 85 wt% of the core particle. The composition of claim 1 , wherein the core particles have an average size of at least 20 nm and no greater than 1 μm. The composition of claim 16 , wherein the average size of the core particles is measured as a Z-average diameter by dynamic light scattering. The composition of claim 1 , wherein the core particles are nanoparticles of a solid drug or a salt thereof. The composition of claim 18 , wherein the average size of the coated nanoparticles is measured as a Z-average diameter by dynamic light scattering. The composition of claim 1 , wherein the coated nanoparticles have a size polydispersity index of 0.5 or less, as measured by dynamic light scattering. The composition of claim 1 , wherein the coated nanoparticles diffuse through the mucus of the human cervix ex vivo with a diffusivity greater than 1/500 of the diffusivity at which the particles diffuse through water on a time scale of 1 second. . 22. The composition of claim 21, wherein diffusion through the mucus is measured ex vivo in mucus of the human cervix. 22. The composition of claim 21, wherein diffusion through mucus is measured in bulk by bulk transport through mucus in the human cervix or microscopic particle tracking. The method of claim 1 , wherein the plurality of coated nanoparticles diffuse or transport through the mucus at a higher rate or greater amount than the plurality of control nanoparticles in the control suspension, wherein the control nanoparticles (i) sodium dodecyl alcohol Pharmaceutical nanoparticles coated non-covalently with a paint or (ii) carboxylated polystyrene nanoparticles without a surface-altering agent coating. 25. The composition of claim 24, wherein diffusion or transport through the mucus is measured ex vivo in human cervical mucus. 25. The composition of claim 24, wherein the transport through mucus is measured in bulk by bulk transport through mucus in the human cervix or by particle tracking microscopically. 27. A pharmaceutical composition comprising the composition of any one of claims 1-26 and one or more pharmaceutically acceptable carriers, additives and/or diluents. 27. A pharmaceutical formulation suitable for inhalation, injection or topical administration across the mucosal barrier, comprising the composition of any one of claims 1-26. 29. The pharmaceutical formulation of claim 28, wherein the coated nanoparticles cross the mucosal barrier. 29. The pharmaceutical formulation of claim 28, wherein the mucosal barrier is mucus. 27. A pharmaceutical composition comprising the composition of any one of claims 1-26 and one or more pharmaceutically acceptable carriers, additives and/or diluents, for delivery of a solid medicament or a salt thereof through a mucosal barrier. 32. The pharmaceutical composition of claim 31, wherein the coated nanoparticles cross the mucosal barrier. 32. The pharmaceutical composition of claim 31, wherein the mucosal barrier is mucus.

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